Nitroxyl donors with improved therapeutic index

ABSTRACT

The disclosed subject matter provides N-substituted hydroxylamine derivative compounds, pharmaceutical compositions and kits comprising such compounds, and methods of using such compounds or pharmaceutical compositions. In particular, the disclosed subject matter provides methods of using such compounds or pharmaceutical compositions for treating heart failure.

1. BACKGROUND

Nitroxyl (HNO) has been shown to have positive cardiovascular effects inin vitro and in vivo models of failing hearts. However, at physiologicalpH, nitroxyl dimerizes to hyponitrous acid, which subsequentlydehydrates to nitrous oxide; due to this metastability, nitroxyl fortherapeutic use must be generated in situ from donor compounds. Avariety of compounds capable of donating nitroxyl have been describedand proposed for use in treating disorders known or suspected to beresponsive to nitroxyl. See, e.g., U.S. Pat. Nos. 6,936,639; 7,696,373;8,030,356; 8,268,890; 8,227,639; and 8,318,705 and U.S. pre-grantpublication nos. 2009/0281067; 2009/0298795; 2011/0136827; and2011/0144067. Although all of these compounds are capable of donatingnitroxyl, they differ in various physicochemical properties, and thereremains a need to identify nitroxyl donors that have physicochemicalproperties best suited for treating specific clinical conditions viaspecific routes of administration.

U.S. Pat. No. 8,030,056 describes the synthesis of derivatives ofPiloty's acid type compounds that are capable of donating nitroxyl underphysiological conditions and are useful in treating heart failure andischemia/reperfusion injury. The nitroxyl donor CXL-1020(N-hydroxy-2-methanesulfonylbenzene-1-sulfonamide) has been evaluated ina Phase I safety study in healthy volunteers and in a Phase IIaplacebo-controlled, double-blind, dose-escalation study conducted atmultiple hospitals. Sabbah et al., “Nitroxyl (HNO) a novel approach forthe acute treatment of heart failure”, Circ Heart Fail., publishedonline Oct. 9, 2013 (Online ISSN: 1941-3297, Print ISSN: 1941-3289). Thestudies demonstrated that in patients with systolic heart failure,CXL-1020, when administered intravenously as an aqueous solution atpH=4, reduced both left and right heart filling pressures and systemicvascular resistance, while increasing cardiac and stroke volume index.Hence, the studies demonstrated that CXL-1020 enhances myocardialfunction in human patients suffering from heart failure. However, atthreshold doses of CXL-1020 needed to produce hemodynamic effects, thecompound was found to induce side effects including unacceptable levelsof inflammatory irritation at and distal to the intravenous insertionsite, and the authors report that because of such side effects, thiscompound would not be a viable candidate for a human therapeutic.

Accordingly, there is a need to develop new nitroxyl donating compoundsand compositions that are useful for the treatment of heart failure andthat have a suitable toxicological profile. Development of suchcompounds requires an understanding of the pharmacokinetic profileassociated with nitroxyl donation and the factors influencing thetoxicological profile. Failure to understand these factors has hamperedthe development of nitroxyl donating compounds for clinical use.

Moreover, formulating nitroxyl donating compounds has proven to be aconsiderable challenge. Many of the current nitroxyl donors areinsoluble in aqueous solutions and/or are insufficiently stable.Solubility and stability problems often preclude the use of suchcompounds in pharmaceutical compositions for parenteral and/or oraladministration. Accordingly, there exists a need to develop compositionscontaining nitroxyl donating compounds for parenteral and/or oraladministration that are sufficiently stable and have favorablepharmacological and toxicological profiles.

Citation of any reference in Section 1 of this application is not to beconstrued as an admission that such reference is prior art to thepresent application.

2. SUMMARY OF THE DISCLOSURE

The present disclosure relates to the discovery of nitroxyl donatingcompounds that are highly efficacious in treating cardiovasculardiseases (e.g., heart failure) and have a suitable toxicologicalprofile.

In a particular embodiment, a nitroxyl donating compound of thedisclosure is a compound of the formula (1):

In another embodiment, a nitroxyl donating compound of the disclosure isa compound of the formula (2):

In another embodiment, the disclosure provides compounds of the formula(3):

wherein R is hydrogen, —(C₁-C₆)alkyl, —(C₂-C₄)alkenyl, phenyl, benzyl,cyclopentyl, cyclohexyl, —(C₅-C₇)heterocycloalkyl, benzyloxy,—O—(C₁-C₆)alkyl, —NH₂, —NH—(C₁-C₄)alkyl, or —N((C₁-C₄)alkyl)₂, whereinsaid —(C₁-C₆)alkyl, —(C₂-C₄)alkenyl, phenyl, benzyl, cyclopentyl,cyclohexyl, —(C₅-C₇)heterocycloalkyl, benzyloxy, —O—(C₁-C₆)alkyl,—NH—(C₁-C₄)alkyl, or —N((C₁-C₄)alkyl)₂ can be unsubstituted orsubstituted with one or more substituents selected from halo,—(C₁-C₆)alkyl, —(C₂-C₄)alkenyl, —(C₂-C₃)alkynyl, -(5- or6-membered)heteroaryl, —O—(C₁-C₆)alkyl, —S—(C₁-C₆)alkyl, —C(halo)₃,—CH(halo)₂, —CH₂(halo), —CN, —NO₂, —NH₂, —NH—(C₁-C₄)alkyl,—N(—(C₁-C₄)alkyl)₂, —C(═O)(C₁-C₄)alkyl, —C(═O)O(C₁-C₄)alkyl,—OC(═O)(C₁-C₄)alkyl, —OC(═O)NH₂, —S(═O)(C₁-C₄)alkyl, or—S(═O)₂(C₁-C₄)alkyl. In particular embodiments, R is methyl, ethyl,benzyl, or phenyl. In particular embodiments, R is methyl or ethyl. Inparticular embodiments, R is methyl. In particular embodiments, R isethyl. In particular embodiments, R is benzyl or phenyl. In particularembodiments, R is benzyl. In particular embodiments, R is phenyl.

In another embodiment, the disclosure provides compounds of formula (4):

wherein R and its embodiments are as defined above with respect to thecompound of formula (3).

Compounds of the disclosure have or are believed to have a highlyfavorable therapeutic index. In particular, compounds of formula (1) andformula (2) have both desirable hemodynamic profiles and toxicologicalprofiles. The toxicological profile of the compounds of formula (1) andformula (2) is significantly improved relative to the clinical candidateCXL-1020. It has been discovered that the favorable toxicologicalprofile of the compounds of formula (1) and formula (2) stems in partfrom the half-lives of the compounds, and the discovery of an optimalrange of half-lives for such nitroxyl donors. The compound of formula(1) has a half-life of approximately 68 minutes when measured in anaerated phosphate buffered saline (PBS) solution at a pH of 7.4, andapproximately 65 minutes when measured in human plasma at a pH of 7.4 inthe presence of an anticoagulant (e.g., heparin or sodium citrate), eachmeasured under conditions specified in Example 4. The compound offormula (2) has a half-life of approximately 50 minutes when measured inan aerated phosphate buffered saline (PBS) solution at a pH of 7.4, andapproximately 37 minutes when measured in human plasma at a pH of 7.4 inthe presence of an anticoagulant (e.g., heparin or sodium citrate), eachmeasured under conditions specified in Example 4.

Moreover, compounds of formula (1) and formula (2) are stable in aqueoussolutions and are highly water soluble; they are, thus, amenable to bothparenteral and oral administration. The compound of formula (1) has anequilibrium solubility in water of greater than 100 mg/mL while thecompound of formula (2) has an equilibrium solubility in water ofapproximately 10 mg/mL (e.g., under conditions specified in Example 5).

Compounds of the disclosure can be used to treat a variety of conditionsthat are responsive to nitroxyl therapy. For instance, a nitroxyldonating compound of the disclosure can be used to treat or prevent theoccurrence of cardiovascular diseases. In particular embodiments, anitroxyl donating compound of the disclosure can be used to treatcardiovascular disease, ischemia/reperfusion injury, pulmonaryhypertension or another condition responsive to nitroxyl therapy. Inother embodiments, a nitroxyl donating compound of the disclosure can beused to treat heart failure. In a particular embodiment, a compound ofthe disclosure can be used to treat decompensated heart failure (e.g.,acute decompensated heart failure). In certain embodiments, thecompounds of the disclosure can be used to treat systolic heart failure.In particular embodiments, the compounds of the disclosure can be usedto treat diastolic heart failure.

In one aspect, the compounds of the disclosure can be administered viaparenteral (e.g., subcutaneous, intramuscular, intravenous orintradermal) administration. The compounds of the disclosure do notinduce undesirable local side effects (e.g., irritation and/orinflammation) during or after parenteral administration at doses capableof providing a desired level of efficacy.

In embodiments in which a compound of the disclosure is administeredparenterally, it is generally administered as an aqueous solution orsuspension. The aqueous solution or suspension can have a pH of fromabout 4 to about 6.5. In particular embodiments, a compound of thedisclosure can be formulated for parenteral injection at a pH of fromabout 4 to about 5. In other embodiments, a compound of the disclosurecan be formulated for parenteral injection at a pH of from about 5 toabout 6. In some embodiments, the formulation for parenteraladministration can include a stability enhancing agent.

When administered parenterally (e.g., intravenously) to a human subject,a compound of the disclosure can be dosed at a rate of from about 5μg/kg/min to about 100 μg/kg/min. In certain embodiments, a compound ofthe disclosure can be dosed to a human subject at a rate of from about10 μg/kg/min to about 70 μg/kg/min. In certain embodiments, a compoundof the disclosure can be dosed to a human subject at a rate of fromabout 15 μg/kg/min to about 50 μg/kg/min. In certain embodiments, acompound of the disclosure can be dosed to a human subject at a rate offrom about 20 μg/kg/min to about 40 μg/kg/min.

In another embodiment, the compounds of the disclosure can be formulatedfor oral administration. Compounds for oral administration can beformulated as liquid or solid dosage forms. In particular embodimentswhere a nitroxyl donating compound is formulated as an oral liquiddosage form, polyethylene glycol 300 (PEG300) can serve as an exemplaryexcipient.

3. BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the hemodynamic profile of CXL-1020 and two compounds ofthe disclosure (compounds of formula (1) and formula (2)) using atachycardia-pacing model of heart failure (see Example 6). Each compoundwas administered intravenously at a rate of 100 μg/kg/min. Hemodynamicparameters were obtained 180 minutes after administration of therespective compound.

FIG. 2 shows the hemodynamic profile of the compound of formula (1) atvarious dosages using a tachycardia-pacing model of heart failure forconscious animals (see Example 6).

FIG. 3 shows the hemodynamic profile of the compound of formula (1)following induction of heart failure in dogs. Hemodynamics wereevaluating using a canine microembolization heart failure model (seeExample 7). The data is shown for final time point during infusion (180minutes) at two rates of infusion.

FIG. 4 shows the assessment of the toxicological profile of CXL-1020 andtwo nitroxyl donating compounds of the disclosure (compounds of formula(1) and formula (2)) following 24 hour infusion at different doses usinga canine peripheral vein model (see Example 9). Key inflammatory markersmeasured include white blood cells (WBC), fibrinogen, and C-reactiveprotein (CRP).

FIG. 5 shows measures of inflammation observed using a canine implantedcentral catheter 72 hour model using different doses of CXL-1020 and thecompounds of formula (1) and (2) (see Example 9).

4. DETAILED DESCRIPTION

The invention includes the following:

(1.) A compound of formula (1):

(2.) A compound of formula (2):

(3.) A pharmaceutical composition comprising a compound of the above(1.) or the above (2.) and at least one pharmaceutically acceptableexcipient.

(4.) The pharmaceutical composition of the above (3.), wherein thepharmaceutical composition is suitable for intravenous administration.

(5.) The pharmaceutical composition of the above (3.) or the above (4.),wherein the pharmaceutical composition has a pH of from about 4 to about6.

(6.) The pharmaceutical composition of any one of the above (3.)-(5.),wherein the pharmaceutical composition has a pH of from about 4 to about5.

(7.) The pharmaceutical composition of any one of the above (3.)-(6.),wherein the pharmaceutical composition has a pH of about 4.

(8.) A method of treating a cardiovascular disease, comprisingadministering an effective amount of compound of the above (1.) or theabove (2.) or the pharmaceutical composition of any one of the above(3.)-(7.) to a patient in need thereof.

(9.) The method of the above (8.), wherein the cardiovascular disease isheart failure.

(10.) The method of the above (8.) or the above (9.), wherein thecardiovascular disease is acute decompensated heart failure.

(11.) The method of any one of the above (8.)-(10.), wherein thecompound or pharmaceutical composition is administered intravenously.

(12.) The method of any one of the above (8.)-(11.), wherein thecompound or pharmaceutical composition is administered at a dose of fromabout 20 μg compound of formula (1) or (2)/kg/minute to about 40 μgcompound of formula (1) or (2)/kg/minute.

(13.) The method of any one of the above (8.)-(10.), wherein thecompound or pharmaceutical composition is administered orally.

(14.) A kit comprising a compound of the above (1.) or the above (2.) indry form or the pharmaceutical composition of any one of the above(3.)-(7.) in dry form; and

a pharmaceutically acceptable liquid diluent.

(15.) Use of the compound of the above (1.) or the above (2.) or use ofthe pharmaceutical composition of any one of the above (3.)-(7.) for themanufacture of a medicament useful for treating a cardiovasculardisease.

(16.) Use of the compound of the above (1.) or the above (2.) or use ofthe pharmaceutical composition of any one of the above (3.)-(7.) for themanufacture of a medicament useful for treating heart failure.

(17.) Use of the compound of the above (1.) or the above (2.) or use ofthe pharmaceutical composition of any one of the above (3.)-(7.) for themanufacture of a medicament useful for treating acute decompensatedheart failure.

(18.) The compound of the above (1.) or the above (2.) or thepharmaceutical composition of any one of the above (3.)-(7.) for use inthe treatment of a cardiovascular disease.

(19.) The compound of the above (1.) or the above (2.) or thepharmaceutical composition of any one of the above (3.)-(7.) for use inthe treatment of heart failure.

(20.) The compound of the above (1.) or the above (2.) or thepharmaceutical composition of any one of the above (3.)-(7.) for use inthe treatment of acute decompensated heart failure.

4.1 Definitions

Unless clearly indicated otherwise, the following terms as used hereinhave the meanings indicated below.

A “pharmaceutically acceptable salt” refers to a salt of any therapeuticagent disclosed herein, which salt can include any of a variety oforganic and inorganic counter ions known in the art and which salt ispharmaceutically acceptable. When the therapeutic agent contains anacidic functionality, various exemplary embodiments of counter ions aresodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, andthe like. When the therapeutic agent contains a basic functionality, apharmaceutically acceptable salt can include as a counter ion, by way ofexample, an organic or inorganic acid, such as hydrochloride,hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and thelike. Illustrative salts include, but are not limited to, sulfate,citrate, acetate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, besylate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, and p-toluenesulfonate salts. Accordingly, a salt canbe prepared from a compound of any one of the formulae disclosed hereinhaving an acidic functional group, such as a carboxylic acid functionalgroup, and a pharmaceutically acceptable inorganic or organic base.Suitable bases include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl-N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower-alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,N,N-di-lower-alkyl-N-(hydroxy-lower-alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl) amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike. A salt can also be prepared from a compound of any one of theformulae disclosed herein having a basic functional group, such as anamino functional group, and a pharmaceutically acceptable inorganic ororganic acid. Suitable acids include hydrogen sulfate, citric acid,acetic acid, hydrochloric acid (HCl), hydrogen bromide (HBr), hydrogeniodide (HI), nitric acid, phosphoric acid, lactic acid, salicylic acid,tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid,fumaric acid, gluconic acid, glucaronic acid, formic acid, benzoic acid,glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid.

“Pharmaceutically acceptable excipient” refers to any substance, notitself a therapeutic agent, used as a carrier, diluent, adjuvant,binder, and/or vehicle for delivery of a therapeutic agent to a patient,or added to a pharmaceutical composition to improve its handling orstorage properties or to permit or facilitate formation of a compound orpharmaceutical composition into a unit dosage form for administration.Pharmaceutically acceptable excipients are known in the pharmaceuticalarts and are disclosed, for example, in Gennaro, Ed., Remington: TheScience and Practice of Pharmacy, 20^(th) Ed. (Lippincott Williams &Wilkins, Baltimore, Md., 2000) and Handbook of PharmaceuticalExcipients, American Pharmaceutical Association, Washington, D.C.,(e.g., 1^(st), 2^(nd) and 3^(rd) Eds., 1986, 1994 and 2000,respectively). As will be known to those in the art, pharmaceuticallyacceptable excipients can provide a variety of functions and can bedescribed as wetting agents, buffering agents, suspending agents,lubricating agents, emulsifiers, disintegrants, absorbents,preservatives, surfactants, colorants, flavorants, and sweeteners.Examples of pharmaceutically acceptable excipients include withoutlimitation: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose,cellulose acetate, hydroxypropylmethylcellulose, andhydroxypropylcellulose; (4) powdered tragacanth; (5) malt; (6) gelatin;(7) talc; (8) excipients, such as cocoa butter and suppository waxes;(9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; and (22) other non-toxic compatible substances employedin pharmaceutical formulations.

“Unit dosage form” refers to a physically discrete unit suitable as aunitary dosage for a human or an animal. Each unit dosage form cancontain a predetermined amount of a therapeutic agent calculated toproduce a desired effect.

Unless clearly indicated otherwise, a “patient” refers to an animal,such as a mammal, including but not limited to, a human. Hence, themethods disclosed herein can be useful in human therapy and veterinaryapplications. In particular embodiments, the patient is a mammal. Incertain embodiments, the patient is a human.

“Effective amount” refers to such amount of a therapeutic agent or apharmaceutically acceptable salt thereof, which in combination with itsparameters of efficacy and potential for toxicity, as well as based onthe knowledge of the practicing specialist, should be effective in agiven therapeutic form. As is understood in the art, an effective amountcan be administered in one or more doses.

“Treatment”, “treating” and the like is an approach for obtaining abeneficial or desired result, including clinical results. For purposesof this disclosure, beneficial or desired results include but are notlimited to inhibiting and/or suppressing the onset and/or development ofa condition or reducing the severity of such condition, such as reducingthe number and/or severity of symptoms associated with the condition,increasing the quality of life of those suffering from the condition,decreasing the dose of other medications required to treat thecondition, enhancing the effect of another medication a patient istaking for the condition, and/or prolonging survival of patients havingthe condition.

“Prevent”, “preventing” and the like refers to reducing the probabilityof developing a condition in a patient who does not have, but is at riskof developing a condition. A patient “at risk” may or may not have adetectable condition, and may or may not have displayed a detectablecondition prior to the treatment methods disclosed herein. “At risk”denotes that a patient has one or more so-called risk factors, which aremeasurable parameters that correlate with development of a condition andare known in the art. A patient having one or more of these risk factorshas a higher probability of developing the condition than a patientwithout such risk factor(s).

“Positive inotrope” refers to an agent that causes an increase inmyocardial contractile function. Exemplary positive inotropes are abeta-adrenergic receptor agonist, an inhibitor of phosphodiesteraseactivity, and calcium-sensitizers. Beta-adrenergic receptor agonistsinclude, among others, dopamine, dobutamine, terbutaline, andisoproterenol. Analogs and derivatives of such compounds are alsointended. For example, U.S. Pat. No. 4,663,351 discloses a dobutamineprodrug that can be administered orally.

A condition that is “responsive to nitroxyl therapy” includes anycondition in which administration of a compound that donates aneffective amount of nitroxyl under physiological conditions treatsand/or prevents the condition, as those terms are defined herein. Acondition whose symptoms are suppressed or diminished uponadministration of nitroxyl donor is a condition responsive to nitroxyltherapy.

“Pulmonary hypertension” or “PH” refers to a condition in which thepulmonary arterial pressure is elevated. The current hemodynamicdefinition of PH is a mean pulmonary arterial pressure (MPAP) at rest ofgreater than or equal to 25 mmHg. Badesch et al., J. Amer. Coll.Cardiol. 54(Suppl.):S55-S66 (2009).

“N/A” means not assessed.

“(C₁-C₆)alkyl” refers to saturated linear and branched hydrocarbonstructures having 1, 2, 3, 4, 5 or 6 carbon atoms. When an alkyl residuehaving a specific number of carbons is named, all geometric isomershaving that number of carbons are intended to be encompassed; thus, forexample, “propyl” includes n-propyl and iso-propyl and “butyl” includesn-butyl, sec-butyl, iso-butyl and tert-butyl. Examples of (C₁-C₆)alkylgroups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl,n-hexyl, and the like.

“(C₁-C₄)alkyl” refers to saturated linear and branched hydrocarbonstructures having 1, 2, 3, or 4 carbon atoms. Examples of (C₁-C₄)alkylgroups include methyl, ethyl, n-propyl, iso-propyl, n-butyl andtert-butyl.

“(C₃-C₅)alkyl” refers to saturated linear and branched hydrocarbonstructures having 3, 4, or 5 carbon atoms. When an alkyl residue havinga specific number of carbons is named, all geometric isomers having thatnumber of carbons are intended to be encompassed; thus, for example,“propyl” includes n-propyl and iso-propyl and “butyl” includes n-butyl,sec-butyl, iso-butyl and tert-butyl. Examples of (C₃-C₅)alkyl groupsinclude n-propyl, iso-propyl, n-butyl, tert-butyl, n-pentyl, and thelike.

“(C₂-C₄)alkenyl” refers to a straight-chain or branched unsaturatedhydrocarbon radical having 2, 3, or 4 carbon atoms and a double bond inany position, e.g., ethenyl, 1-propenyl, 2-propenyl (allyl), 1-butenyl,2-butenyl, 3-butenyl, 1-methylethenyl, 1-methyl-1-propenyl,2-methyl-2-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, and thelike.

“(C₂-C₃)alkynyl” refers to a straight chain non-cyclic hydrocarbonhaving 2 or 3 carbon atoms and including at least one carbon-carbondouble bond. Examples of (C₂-C₃)alkenyls include -vinyl, -allyl, and1-prop-1-enyl.

“(C₅-C₇)heterocycloalkyl” refers to a 5-, 6-, or 7-membered, saturatedor unsaturated, bridged, mono- or bicyclic-heterocycle containing 1, 2,3, or 4 ring heteroatoms each independently selected from nitrogen,oxygen, and sulfur. Examples of (C₅-C₇)heterocycloalkyl groups includepyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydro-oxazinyl,tetrahydrofuran, thiolane, dithiolane, pyrroline, pyrrolidine,pyrazoline, pyrazolidine, imidazoline, imidazolidine, tetrazole,piperidine, pyridazine, pyrimidine, pyrazine, tetrahydrofuranone,γ-butyrolactone, α-pyran, γ-pyran, dioxolane, tetrahydropyran, dioxane,dihydrothiophene, piperazine, triazine, tetrazine, morpholine,thiomorpholine, diazepan, oxazine, tetrahydro-oxazinyl, isothiazole,pyrazolidine, and the like.

“(5- or 6-membered)heteroaryl” refers to a monocyclic aromaticheterocycle ring of 5 or 6 members, i.e., a monocyclic aromatic ringcomprising at least one ring heteroatom, e.g., 1, 2, 3, or 4 ringheteroatoms, each independently selected from nitrogen, oxygen, andsulfur. Examples of -(5- or 6-membered)heteroaryls include pyridyl,pyrrolyl, furyl, imidazolyl, oxazolyl, imidazolyl, thiazolyl,isoxazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,3-triazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidyl,pyrazinyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,5-triazinyl, and thiophenyl.

“Halo” refers to —F, —Cl, —Br or —I.

“Sulfo-n-butyl ether derivative of β-cyclodextrin” refers toβ-cyclodextrin having at least one —OH group that is derivatized byreplacing the hydrogen atom thereof with —(CH₂)₄—S(O)₂—OH or—(CH₂)₄—S(O)₂—O⁻Z⁺ to provide a —O—(CH₂)₄—S(O)₂—OH or—O—(CH₂)₄—S(O)₂—O⁻Z⁺ group, respectively, where Z⁺ is a cation such assodium, potassium, ammonium, tetramethylammonium, and the like. In oneembodiment, each Z is sodium.

4.2 Nitroxyl Donating Compounds with Improved Therapeutic Index

In one aspect, the disclosure provides novel compounds suitable fortreating cardiovascular diseases (e.g., heart failure). In particular,the disclosure provides nitroxyl donating compounds that have acombination of properties that make them suitable for use as a humantherapeutic. In particular, the nitroxyl donating compounds of thedisclosure have suitable half-lives, a favorable therapeutic index, arehighly water soluble and have sufficient solid state stability. Table 1provides two specific N-hydroxysulfonamide nitroxyl donating compoundsof the disclosure that possess such desirable properties and are thussuitable for therapeutic administration to humans.

TABLE 1 Nitroxyl Donating Compounds of the Disclosure

(1) N-Hydroxy-5-methylfuran-2-sulfonamide

(2) N-Hydroxy-3-methanesulfonylbenzene-1-sulfonamide

In particular embodiments, the nitroxyl donating compounds in Table 1can be utilized as a pharmaceutically acceptable salt thereof.

In other embodiments, the N-hydroxy group of the compounds listed inTable 1 can be esterified to provide prodrugs of the compounds.

For instance, the disclosure provides compounds of the formula (3):

wherein R is hydrogen, —(C₁-C₆)alkyl, —(C₂-C₄)alkenyl, phenyl, benzyl,cyclopentyl, cyclohexyl, —(C₅-C₇)heterocycloalkyl, benzyloxy,—O—(C₁-C₆)alkyl, —NH₂, —NH—(C₁-C₄)alkyl, or —N((C₁-C₄)alkyl)₂, whereinsaid —(C₁-C₆)alkyl, —(C₂-C₄)alkenyl, phenyl, benzyl, cyclopentyl,cyclohexyl, —(C₅-C₇)heterocycloalkyl, benzyloxy, —NH—(C₁-C₄)alkyl, or—N((C₁-C₄)alkyl)₂ can be unsubstituted or substituted with one or moresubstituents selected from halo, —(C₁-C₆)alkyl, —(C₂-C₄)alkenyl,—(C₂-C₃)alkynyl, -(5- or 6-membered)heteroaryl, —O—(C₁-C₆)alkyl,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —CN, —NO₂, —NH₂, —NH—(C₁-C₄)alkyl,—N(—(C₁-C₄)alkyl)₂, —C(═O)(C₁-C₄)alkyl, —C(═O)O(C₁-C₄)alkyl,—OC(═O)(C₁-C₄)alkyl, —OC(═O)NH₂, —S(═O)(C₁-C₄)alkyl, or—S(═O)₂(C₁-C₄)alkyl. In particular embodiments, R is methyl, ethyl,benzyl, or phenyl.

In particular embodiments where the compound of the disclosure is acompound of the formula (3), R is methyl. In other embodiments where thecompound has the formula (3), R is ethyl. In certain embodiments wherethe compound of the disclosure is a compound of the formula (3), R ismethyl or ethyl. In other embodiments where the compound where thecompound has the formula (3), R is phenyl. In other embodiments wherethe compound has the formula (3), R is benzyl. In particular embodimentswhere the compound of the disclosure is a compound of the formula (3), Ris benzyl or phenyl. In other embodiments where the compound has theformula (3), R is —NH₂. In each of the above embodiments in thisparagraph, R is unsubstituted in one embodiment, mono-substituted inanother embodiment, di-substituted with two independently selectedsubstituents in an additional embodiment, or tri-substituted with threeindependently selected substituents in a further embodiment. In variousembodiments of each of the above embodiments in this paragraph, thesubstituent is -halo, —NH₂, —NHCH₃, —CF₃, or —OCH₃ or the substituentsare independently selected from -halo, —NH₂, —NHCH₃, —CF₃, and —OCH₃.

For instance, the disclosure provides compounds of the formula (4):

wherein R and its optional substituent(s) are as defined above withrespect to the compound of formula (3).

In particular embodiments where the compound of the disclosure is acompound of the formula (4), R is methyl. In other embodiments where thecompound has the formula (4), R is ethyl. In certain embodiments wherethe compound of the disclosure is a compound of the formula (4), R ismethyl or ethyl. In other embodiments where the compound where thecompound has the formula (4), R is phenyl. In other embodiments wherethe compound has the formula (4), R is benzyl. In particular embodimentswhere the compound of the disclosure is a compound of the formula (4), Ris benzyl or phenyl. In other embodiments where the compound has theformula (4), R is —NH₂. In each of the above embodiments in thisparagraph, R is unsubstituted in one embodiment, mono-substituted inanother embodiment, di-substituted with two independently selectedsubstituents in an additional embodiment, or tri-substituted with threeindependently selected substituents in a further embodiment. In variousembodiments of each of the above embodiments in this paragraph, thesubstituent is -halo, —NH₂, —NHCH₃, —CF₃, or —OCH₃ or the substituentsare independently selected from -halo, —NH₂, —NHCH₃, —CF₃, and —OCH₃.

Unexpectedly, it has been discovered that the nitroxyl donatingcompounds of the disclosure provide levels of efficacy similar toCXL-1020 when administered to human patients, but with significantlyreduced side effects, notably local side effects (e.g., irritationand/or inflammation) (see Examples 8 and 9). Moreover, nitroxyl donatingcompounds of the disclosure provide an onset of hemodynamic effects in 1hour or less, which is desirable from a clinical perspective.

Without being bound by theory, the experiments reported in the Examplesof this disclosure suggest that nitroxyl donors with half-livessubstantially shorter than 15 minutes when measured in PBS or humanplasma (see Example 4), such as CXL-1020, produce high localconcentrations of nitroxyl upon administration, and that the high localconcentration of nitroxyl is a cause of the observed undesirable sideeffects. Nitroxyl at high concentration is known to dimerize, resultingin the formation of hyponitrous acid, which is capable of producinghydroxyl radicals. Alternatively, or in addition, peroxide emanatingfrom white blood cells can react with nitroxyl to form hydroxylradicals. Hydroxyl radicals can be toxic to endothelial cells, resultingin inflammation and/or intolerance. While nitroxyl compounds with longerhalf-lives could, in theory, produce hydroxyl radicals through similarmechanisms, formation of such radicals would be expected to be reducedby virtue of the low concentrations of nitroxyl, thus reducing theability of nitroxyl to dimerize or to react with peroxide. Compoundswith very long half-lives (e.g., greater than 95 minutes when measuredin human plasma in accordance with the method described in Example 4)would therefore be expected to have a favorable toxicological profile;however, because these compounds would be expected to be cleared fromthe circulation and/or diluted prior to substantial nitroxyl formation,such compounds are expected to have low efficacy.

As described in Example 4, compounds of formulas (1) and (2) havehalf-lives greater than about 10 minutes and less than 95 minutes whenmeasured in an aerated phosphate buffered saline (PBS) solution at a pHof 7.4, and when measured in human plasma at pH 7.4 in the presence ofan anticoagulant (e.g., heparin or sodium citrate), each measured underconditions specified in Example 4. In particular, the compound offormula (1) has a half-life of approximately 68 minutes when measured inan aerated phosphate buffered saline (PBS) solution at a pH of 7.4, andapproximately 65 minutes when measured in human plasma at pH 7.4 in thepresence of an anticoagulant (e.g., heparin or sodium citrate), eachmeasured under conditions specified in Example 4. The compound offormula (2) has a half-life of approximately 50 minutes when measured inan aerated phosphate buffered saline (PBS) solution at a pH of 7.4, andapproximately 37 minutes when measured in human plasma at pH 7.4 in thepresence of an anticoagulant (e.g., heparin or sodium citrate), eachmeasured under conditions specified in Example 4.

Furthermore, as described in Example 5, each of the compounds offormulas (1) and (2) is highly water soluble and is thus amenable toparenteral or oral administration. The compounds can be formulatedwithout the addition of a solubilizing agent. Moreover, as demonstratedin Examples 10-12, the compounds of formula (1) and formula (2) haveexcellent stability in pharmaceutical compositions for parenteral (e.g.,intravenous) administration.

4.3 Measuring Nitroxyl Donating Ability

Compounds are easily tested for nitroxyl donation by routineexperiments. Although it is typically impractical to directly measurewhether nitroxyl is donated, several analytical approaches are acceptedas suitable for determining whether a compound donates nitroxyl. Forexample, the compound of interest can be placed in solution, for examplein phosphate buffered saline (PBS) or in a phosphate buffered solutionat a pH of about 7.4, in a sealed container. After sufficient time fordisassociation has elapsed, such as from several minutes to severalhours, the headspace gas is withdrawn and analyzed to determine itscomposition, such as by gas chromatography and/or mass spectrometry. Ifthe gas N₂O is formed (which occurs by HNO dimerization), the test ispositive for nitroxyl donation and the compound is deemed to be anitroxyl donor.

The level of nitroxyl donating ability can be expressed as a percentageof a compound's theoretical stoichiometric maximum. A compound thatdonates a “significant level of nitroxyl” means, in various embodiments,a compound that donates about 40% or more, about 50% or more, about 60%or more, about 70% or more, about 80% or more, about 90% or more, orabout 95% or more of its theoretical maximum amount of nitroxyl. Inparticular embodiments, a nitroxyl donor of the disclosure compoundherein donates from about 70% to about 90% of its theoretical maximumamount of nitroxyl. In particular embodiments, a nitroxyl donor of thedisclosure compound herein donates from about 85% to about 95% of itstheoretical maximum amount of nitroxyl. In particular embodiments, anitroxyl donor of the disclosure compound herein donates from about 90%to about 95% of its theoretical maximum amount of nitroxyl. Compoundsthat donate less than about 40%, or less than about 50%, of theirtheoretical maximum amount of nitroxyl are still nitroxyl donors and canbe used in the methods disclosed. A compound that donates less thanabout 50% of its theoretical amount of nitroxyl can be used in themethods disclosed, but may require higher dosing levels as compared to acompound that donates a higher level of nitroxyl.

If desired, nitroxyl donation also can be detected by exposing the testcompound to metmyoglobin (Mb³⁺). See Bazylinski et al., J. Amer. Chem.Soc. 107(26):7982-7986 (1985). Nitroxyl reacts with Mb³⁺ to form aMb²⁺—NO complex, which can be detected by changes in theultraviolet/visible spectrum or by electron paramagnetic resonance(EPR). The Mb²⁺—NO complex has an EPR signal centered around a g-valueof about 2. Nitric oxide, on the other hand, reacts with Mb³⁺ to form anMb³⁺—NO complex that has a negligible, if any, EPR signal. Accordingly,if a compound reacts with Mb³⁺ to form a complex detectable by commonmethods, such as ultraviolet/visible or EPR, then the test is positivefor nitroxyl donation.

Testing for nitroxyl donation can be performed at a physiologicallyrelevant pH. The nitroxyl donating compounds of the disclosure arecapable of donating nitroxyl at physiological pH (i.e., a pH of about7.4) and physiological temperature (i.e., a temperature of about 37° C.)(together, “physiological conditions”). In particular embodiments, anitroxyl donating compound of the disclosure can donate about 40% ormore of its theoretical maximum (i.e., 100%) amount of nitroxyl underphysiological conditions. In particular embodiments, a nitroxyl donatingcompound of the disclosure can donate about 50% or more of itstheoretical maximum amount of nitroxyl under physiological conditions.In particular embodiments, a nitroxyl donating compound of thedisclosure can donate about 60% or more of its theoretical maximumamount of nitroxyl under physiological conditions. In particularembodiments, a nitroxyl donating compound of the disclosure can donateabout 70% or more of its theoretical maximum amount of nitroxyl underphysiological conditions. In particular embodiments, a nitroxyl donatingcompound of the disclosure can donate about 80% or more of itstheoretical maximum amount of nitroxyl under physiological conditions.In particular embodiments, a nitroxyl donating compound of thedisclosure can donate about 90% or more of its theoretical maximumamount of nitroxyl under physiological conditions.

It will be understood that a nitroxyl donating compound of thedisclosure might also donate a limited amount of nitric oxide, so longas the amount of nitroxyl donation exceeds the amount of nitric oxidedonation. In certain embodiments, a nitroxyl donating compound candonate about 25 mole % or less of nitric oxide under physiologicalconditions. In particular embodiments, a nitroxyl donating compound candonate about 20 mole % or less of nitric oxide under physiologicalconditions. In particular embodiments, a nitroxyl donating compound candonate about 15 mole % or less of nitric oxide under physiologicalconditions. In particular embodiments, a nitroxyl donating compound candonate about 10 mole % or less of nitric oxide under physiologicalconditions. In particular embodiments, a nitroxyl donating compound candonates about 5 mole % or less of nitric oxide under physiologicalconditions. In particular embodiments, a nitroxyl donating compound candonate about 2 mole % or less of nitric oxide under physiologicalconditions. In particular embodiments, a nitroxyl donating compound candonate an insignificant amount (e.g., about 1 mole % or less) of nitricoxide under physiological conditions.

4.4 Pharmaceutical Compositions

The disclosure also encompasses pharmaceutical compositions comprising anitroxyl donating compound of formulae (1), (2), (3), or (4) and atleast one pharmaceutically acceptable excipient. Examples ofpharmaceutically acceptable excipients include those described above,such as carriers, surface active agents, thickening or emulsifyingagents, solid binders, dispersion or suspension aids, solubilizers,colorants, flavoring agents, coatings, disintegrating agents,lubricants, sweeteners, preservatives, isotonic agents, and anycombination thereof. The selection and use of pharmaceuticallyacceptable excipients is taught, e.g., in Troy, Ed., Remington: TheScience and Practice of Pharmacy, 21^(st) Ed. (Lippincott Williams &Wilkins, Baltimore, Md., 2005).

In various embodiments, the at least one pharmaceutically acceptableexcipient comprises at least one species of cyclodextrin. In aparticular embodiment, the cyclodextrin is a cyclic structure havingglucose units linked by α(1-4) linkages. In another embodiment, thecyclodextrin is a β-cyclodextrin, i.e., a cyclic structure having sevenglucose units linked by α(1-4) linkages. In another embodiment, thecyclodextrin is chemically modified by derivatizing any combination ofthe three available hydroxyl groups on each glucopyranose unit thereof.

In some embodiments where the pharmaceutically acceptable excipientcomprises at least one species of cyclodextrin, the cyclodextrin is asulfo(C₁-C₆)alkyl ether derivative of β-cyclodextrin. In certain ofthese embodiments, the cyclodextrin is a sulfo(C₁-C₆)alkyl etherderivative of β-cyclodextrin having from about six to about sevensulfo(C₁-C₆)alkyl ether groups per cyclodextrin molecule. In variousembodiments, the cyclodextrin is a sulfo(C₁-C₆)alkyl ether derivative ofβ-cyclodextrin having an average of from about six to about sevensulfo(C₁-C₆)alkyl ether groups per cyclodextrin molecule. In anothersuch embodiment, the cyclodextrin is a sulfo(C₁-C₆)alkyl etherderivative of β-cyclodextrin having six or seven sulfo(C₁-C₆)alkyl ethergroups per cyclodextrin molecule.

In a particular series of embodiments where the pharmaceuticallyacceptable excipient comprises at least one species of cyclodextrin, thecyclodextrin is a sulfo(C₃-C₅)alkyl ether derivative of β-cyclodextrin.In one such embodiment, the cyclodextrin is a sulfo(C₃-C₅)alkyl etherderivative of β-cyclodextrin having from about six to about sevensulfo(C₃-C₅)alkyl ether groups per cyclodextrin molecule. In varioussuch embodiments, the cyclodextrin is a sulfo(C₃-C₅)alkyl etherderivative of β-cyclodextrin having an average of from about six toabout seven sulfo(C₃-C₅)alkyl ether groups per cyclodextrin molecule. Inanother such embodiment, the cyclodextrin is a sulfo(C₃-C₅)alkyl etherderivative of β-cyclodextrin having six or seven sulfo(C₃-C₅)alkyl ethergroups per cyclodextrin molecule.

In particular embodiments where the pharmaceutically acceptableexcipient comprises at least one species of cyclodextrin, thecyclodextrin is a sulfobutyl ether derivative of 3-cyclodextrin. Incertain of these embodiments, the cyclodextrin is a sulfobutyl etherderivative of β-cyclodextrin having from about six to about sevensulfobutyl ether groups per cyclodextrin molecule. In another suchembodiment, the cyclodextrin is a sulfobutyl ether derivative ofβ-cyclodextrin having an average of from about six to about sevensulfobutyl ether groups per cyclodextrin molecule. In another suchembodiment, the cyclodextrin is a sulfobutyl ether derivative ofβ-cyclodextrin having six or seven sulfobutyl ether groups percyclodextrin molecule.

In certain embodiments where the pharmaceutically acceptable excipientcomprises at least one species of cyclodextrin, the cyclodextrin is asulfo-n-butyl ether derivative of β-cyclodextrin. In one suchembodiment, the cyclodextrin is a sulfo-n-butyl ether derivative ofβ-cyclodextrin having from about six to about seven sulfo-n-butyl ethergroups per cyclodextrin molecule. In another such embodiment, thecyclodextrin is a sulfo-n-butyl ether derivative of β-cyclodextrinhaving an average of from about six to about seven sulfo-n-butyl ethergroups per cyclodextrin molecule. In another such embodiment, thecyclodextrin is a sulfo-n-butyl ether derivative of β-cyclodextrinhaving six or seven sulfo-n-butyl ether groups per cyclodextrinmolecule.

In various particular embodiments where the pharmaceutically acceptableexcipient comprises at least one species of cyclodextrin, thecyclodextrin comprises a plurality of negative charges atphysiologically compatible pH values, e.g., at a pH of from about 5.0 toabout 6.8 in some embodiments, from about 5.5 to about 6.5 in someembodiments, from about 5.7 to about 6.3 in some embodiments, from about5.8 to about 6.2 in some embodiments, from about 5.9 to about 6.1 insome embodiments, and about 6.0 in particular embodiments. In one suchembodiment, the at least one pharmaceutically acceptable excipientcomprises CAPTISOL® cyclodextrin (Ligand Pharmaceuticals, La Jolla,Calif.).

The pharmaceutical compositions can be formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, as drenches (for example, aqueous ornon-aqueous solutions or suspensions), tablets (for example, thosetargeted for buccal, sublingual and systemic absorption), caplets,boluses, powders, granules, pastes for application to the tongue, hardgelatin capsules, soft gelatin capsules, mouth sprays, troches,lozenges, pellets, syrups, suspensions, elixirs, liquids, emulsions andmicroemulsions; or (2) parenteral administration by, for example,subcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension. The pharmaceuticalcompositions can be for immediate, sustained or controlled release.

In one particular embodiment, the pharmaceutical composition isformulated for intravenous administration. In another embodiment, thepharmaceutical composition is formulated for intravenous administrationby continuous infusion.

In another embodiment, the pharmaceutical composition is formulated fororal administration. Compounds for oral administration can be formulatedas liquid or solid dosage forms. In particular embodiments where thenitroxyl donating compounds are formulated as oral liquid dosage forms,polyethylene glycol 300 (PEG300) can usefully serve as an excipient.

The compounds and pharmaceutical compositions disclosed herein can beprepared as any appropriate unit dosage form, such as capsules, sachets,tablets, powder, granules, solution, suspension in an aqueous liquid,suspension in a non-aqueous liquid, oil-in-water liquid emulsion,water-in-oil liquid emulsion, liposomes or bolus.

Tablets can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared bycompressing in a suitable machine the therapeutic agent or agents in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets can be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets can be optionally coated or scored and canbe formulated so as to provide slow or controlled release of the activeingredient therein. Methods of formulating such slow or controlledrelease compositions of pharmaceutically active ingredients, such as thetherapeutic agents herein and other compounds known in the art, areknown in the art and disclosed in issued U.S. patents, some of whichinclude, but are not limited to, U.S. Pat. Nos. 4,369,174, 4,842,866,and the references cited therein. Coatings can be used for delivery ofcompounds to the intestine (see, e.g., U.S. Pat. Nos. 6,638,534,5,217,720, 6,569,457, and the references cited therein). An artisan willrecognize that in addition to tablets, other dosage forms can beformulated to provide slow or controlled release of the activeingredient. Such dosage forms include, but are not limited to, capsules,granulations and gel-caps.

Pharmaceutical compositions suitable for topical administration include,without limitation, lozenges comprising the ingredients in a flavoredbasis, such as sucrose, acacia and tragacanth; and pastilles comprisingthe active ingredient in a flavored basis or in an inert basis, such asgelatin and glycerin.

Various embodiments of pharmaceutical compositions suitable forparenteral administration include, without limitation, either aqueoussterile injection solutions or non-aqueous sterile injection solutions,each containing, for example, anti-oxidants, buffers, bacteriostats andsolutes that render the formulation isotonic with the blood of theintended recipient; and aqueous sterile suspensions and non-aqueoussterile suspensions, each containing, for example, suspending agents andthickening agents. The formulations can be presented in unit-dose ormulti-dose containers, for example, sealed ampules or vials, and can bestored in a freeze dried (lyophilized) condition requiring only theaddition of a sterile liquid carrier, such as water, immediately priorto use.

Pharmaceutical compositions administered parenterally can beadministered in an acidic, neutral or basic solution. In one embodiment,pharmaceutical compositions comprising a nitroxyl donating compound ofthe disclosure can be formulated in an acidic solution having a pH offrom about 4 to about 5, for instance, a pH of about 4, about 4.5, about4.8, or about 5, including values there between. While a pH of about 4has generally been considered optimal for formulating nitroxyl donatingcompositions to achieve adequate stability of the compound, it has beendiscovered that formulating under such acidic conditions can potentiallycause or exacerbate venous irritation following parenteraladministration. The amount of irritation can be attenuated byformulating the nitroxyl donating compounds in less acidic or evenneutral solutions (see FIG. 4). Accordingly, in particular embodiments,a nitroxyl donating compounds of the disclosure can be formulated forparenteral use at a pH of from about 5 to about 6.2 (e.g., pH of about5, about 5.5, about 5.8, about 6, or about 6.2, including values therebetween).

4.5 Methods of Using the Compounds and Pharmaceutical Compositions ofthe Disclosure

In one aspect, the disclosure provides a method of increasing in vivonitroxyl levels, comprising administering to a patient in need thereofan effective amount of a compound or a pharmaceutical composition asdisclosed herein. In various embodiments, the patient has, is suspectedof having, or is at risk of having or developing a condition that isresponsive to nitroxyl therapy.

In particular embodiments, the disclosure provides a method of treating,preventing or delaying the onset and/or development of a condition,comprising administering to a patient (including a patient identified asin need of such treatment, prevention or delay) an effective amount of acompound or a pharmaceutical composition as disclosed herein.Identifying a patient in need thereof can be in the judgment of aphysician, clinical staff, emergency response personnel or other healthcare professional and can be subjective (e.g., opinion) or objective(e.g., measurable by a test or diagnostic method).

Particular conditions embraced by the methods disclosed herein include,without limitation, cardiovascular diseases, ischemia/reperfusioninjury, and pulmonary hypertension (PH).

4.5.1 Cardiovascular Diseases

In one embodiment, the disclosure provides a method of treating acardiovascular disease, comprising administering an effective amount ofa compound or a pharmaceutical composition as disclosed herein to apatient in need thereof.

Examples of cardiovascular diseases and symptoms that can usefully betreated with the compounds and compositions disclosed herein includecardiovascular diseases that are responsive to nitroxyl therapy,coronary obstructions, coronary artery disease (CAD), angina, heartattack, myocardial infarction, high blood pressure, ischemiccardiomyopathy and infarction, pulmonary congestion, pulmonary edema,cardiac fibrosis, valvular heart disease, pericardial disease,circulatory congestive states, peripheral edema, ascites, Chagas'disease, ventricular hypertrophy, heart valve disease, heart failure,diastolic heart failure, systolic heart failure, congestive heartfailure, acute congestive heart failure, acute decompensated heartfailure, and cardiac hypertrophy.

4.5.1.1 Heart Failure

The nitroxyl donating compounds and compositions of the disclosure canbe used to treat patients suffering from heart failure. The heartfailure can be of any type or form, including any of the heart failuresdisclosed herein. Nonlimiting examples of heart failure include earlystage heart failure, Class I, II, III and IV heart failure, acute heartfailure, congestive heart failure (CHF) and acute congestive heartfailure. In one embodiment, the compounds and compositions of thedisclosure can be used to treat acute decompensated heart failure.

In embodiments where the nitroxyl donating compounds and compositions ofthe disclosure are used to treat patients suffering from heart failure,another active agent that treats heart failure can also be administered.In one such embodiment, the nitroxyl donor can be administered inconjunction with a positive inotrope such as a beta-agonist. Examples ofbeta-agonists include, without limitation, dopamine, dobutamine,isoproterenol, analogs of such compounds and derivatives of suchcompounds. In another embodiment, nitroxyl donor can be administered inconjunction with a beta-adrenergic receptor antagonist (also referred toherein as beta-antagonist or beta-blocker). Examples of beta-antagonistsinclude, without limitation, propranolol, metoprolol, bisoprolol,bucindolol, and carvedilol.

As described in Examples 6 and 7, various heart failure models were usedto evaluate the hemodynamic profiles of several of the nitroxyl donatingcompounds of the disclosure. As shown in FIGS. 1-3, which are discussedin Examples 6 and 7, the compounds of formula (1) and formula (2)produced, for example, significant enhancement of inotropy andlusitropy, and modest reductions in blood pressure without tachycardia.Moreover, the onset of significant hemodynamic effects was rapid (e.g.,within 1 hour) and for near-maximal effect was achieved within 2 hours.

While the hemodynamic activity of compounds of formula (1) and formula(2) are similar to compositions comprising the nitroxyl donor CXL-1020when administered intravenously, the toxicological profile of compoundsof formula (1) and formula (2), which have longer half-lives thanCXL-1020, is significantly improved as compared to compositionscomprising CXL-1020 (see Example 9 and FIGS. 4 and 5). For example, the“No Observed Adverse Effect Levels” (NOAEL) of the compounds of formula(1) and formula (2) were substantially higher than the NOAEL forCXL-1020 (see Example 9 for description of NOAEL determination). Inparticular, the compound of formula (1) has the most favorabletoxicological profile of all N-hydroxysulfonamide type nitroxyl donorstested thus far and shows no adverse effects on clinical markers ofinflammation when administered intravenously at concentrations at leastas high as 30 μg/kg/min (FIG. 4). In contrast, CXL-1020 begins to showundesirable side effects at concentrations as low as 0.3 μg/kg/min.

4.5.1.2 Ischemia/Reperfusion Injury

In another embodiment, the disclosed subject matter provides a method oftreating, preventing or delaying the onset and/or development ofischemia/reperfusion injury, comprising administering an effectiveamount of a compound or pharmaceutical composition as disclosed hereinto a subject in need thereof.

In a particular embodiment, the method is for preventingischemia/reperfusion injury. In a particular embodiment, a compound orpharmaceutical composition of the disclosure is administered prior tothe onset of ischemia. In a particular embodiment, a pharmaceuticalcomposition of the disclosure is administered prior to procedures inwhich myocardial ischemia can occur, for example an angioplasty orsurgery, such as a coronary artery bypass graft surgery. In a particularembodiment, a pharmaceutical composition of the disclosure isadministered after ischemia but before reperfusion. In a particularembodiment, a pharmaceutical composition of the disclosure isadministered after ischemia and reperfusion.

In another embodiment, a pharmaceutical composition of the disclosurecan be administered to a patient who is at risk for an ischemic event.In a particular embodiment, a pharmaceutical composition of thedisclosure is administered to a patient at risk for a future ischemicevent, but who has no present evidence of ischemia. The determination ofwhether a patient is at risk for an ischemic event can be performed byany method known in the art, such as by examining the patient or thepatient's medical history. In a particular embodiment, the patient hashad a prior ischemic event. Thus, the patient can be at risk of a firstor subsequent ischemic event. Examples of patients at risk for anischemic event include patients with known hypercholesterolemia, EKGchanges associated with ischemia (e.g., peaked or inverted T-waves or STsegment elevations or depression in an appropriate clinical context),abnormal EKG not associated with active ischemia, elevated CKMB,clinical evidence of ischemia (e.g., crushing sub-sternal chest pain orarm pain, shortness of breath and/or diaphoresis), prior history ofmyocardial infarction, elevated serum cholesterol, sedentary lifestyle,angiographic evidence of partial coronary artery obstruction,echocardiographic evidence of myocardial damage, or any other evidenceof a risk for a future ischemic event. Examples of ischemic eventsinclude, without limitation, myocardial infarction (MI) andneurovascular ischemia, such as a cerebrovascular accident (CVA).

In another embodiment, the subject of treatment is an organ that is tobe transplanted. In a particular embodiment, a pharmaceuticalcomposition of the disclosure can be administered prior to reperfusionof the organ in a transplant recipient. In a particular embodiment, apharmaceutical composition of the disclosure can be administered priorto removal of the organ from the donor, for example through theperfusion cannulas used in the organ removal process. If the organ donoris a live donor, for example a kidney donor, the compounds orpharmaceutical compositions of the disclosure can be administered to theorgan donor. In a particular embodiment, the compounds or pharmaceuticalcompositions of the disclosure are administered by storing the organ ina solution comprising the compound or pharmaceutical composition. Forexample, a compound or pharmaceutical composition of the disclosure canbe included in the organ preservation solution, such as the Universityof Wisconsin “UW” solution, which is a solution comprising hydroxyethylstarch substantially free of ethylene glycol, ethylene chlorohydrin andacetone (see U.S. Pat. No. 4,798,824). In a particular embodiment, apharmaceutical composition of the disclosure that is administered issuch that ischemia/reperfusion injury to the tissues of the organ isreduced upon reperfusion in the recipient of transplanted organ. In aparticular embodiment, the method reduces tissue necrosis (the size ofinfarct) in at-risk tissues.

Ischemia/reperfusion injury can damage tissues other than those of themyocardium and the disclosed subject matter embraces methods of treatingor preventing such damage. In various embodiments, theischemia/reperfusion injury is non-myocardial. In particularembodiments, the method reduces injury from ischemia/reperfusion in thetissue of the brain, liver, gut, kidney, bowel, or any part of the bodyother than the myocardium. In another embodiment, the patient is at riskfor such injury. Selecting a person at risk for non-myocardial ischemiacould include a determination of the indicators used to assess risk formyocardial ischemia. However, other factors can indicate a risk forischemia/reperfusion in other tissues. For example, surgery patientsoften experience surgery related ischemia. Thus, patients scheduled forsurgery could be considered at risk for an ischemic event. The followingrisk factors for stroke (or a subset of these risk factors) coulddemonstrate a patient's risk for ischemia of brain tissue: hypertension,cigarette smoking, carotid artery stenosis, physical inactivity,diabetes mellitus, hyperlipidemia, transient ischemic attack, atrialfibrillation, coronary artery disease, congestive heart failure, pastmyocardial infarction, left ventricular dysfunction with mural thrombus,and mitral stenosis. Ingall, Postgrad. Med. 107(6):34-50 (2000).Further, complications of untreated infectious diarrhea in the elderlycan include myocardial, renal, cerebrovascular and intestinal ischemia.Slotwiner-Nie et al., Gastroenterol. Clin. N. Amer. 30(3):625-635(2001). Alternatively, patients could be selected based on risk factorsfor ischemic bowel, kidney and/or liver disease. For example, treatmentwould be initiated in elderly patients at risk of hypotensive episodes(such as surgical blood loss). Thus, patients presenting with such anindication would be considered at risk for an ischemic event. In anotherembodiment, the patient has any one or more of the conditions listedherein, such as diabetes mellitus and hypertension. Other conditionsthat can result in ischemia, such as cerebral arteriovenousmalformation, could demonstrate a patient's risk for an ischemic event.

4.5.2 Pulmonary Hypertension

In another embodiment, a compounds or pharmaceutical composition of thedisclosure can be used to prevent or delay the onset and/or developmentof pulmonary hypertension. In one such embodiment, a compounds orpharmaceutical composition of the disclosure can be used to prevent ordelay the onset and/or development of pulmonary arterial hypertension(PAH).

In another embodiment, the disclosed subject matter provides a method ofreducing mean pulmonary arterial pressure (MPAP), comprisingadministering an effective amount of a compound or a pharmaceuticalcomposition disclosed herein to a patient in need thereof. In anotherembodiment, the MPAP is reduced by up to about 50%. In anotherembodiment, the MPAP is reduced by up to about 25%. In anotherembodiment, the MPAP is reduced by up to about 20%. In anotherembodiment, the MPAP is reduced by up to about 15%. In anotherembodiment, the MPAP is reduced by up to 10%. In another embodiment, theMPAP is reduced by up to about 5%. In another embodiment, the MPAP isreduced to be from about 12 mmHg to about 16 mmHg. In anotherembodiment, the MPAP is reduced to be about 15 mmHg.

4.6 Administration Modes, Regimens and Dose Levels

The compounds and pharmaceutical compositions of the disclosure can beadministered via parenteral (e.g., subcutaneous, intramuscular,intravenous or intradermal) administration. In certain embodiments, thecompound or pharmaceutical composition is administered by intravenousinfusion. In other embodiments, the compounds and pharmaceuticalcompositions of the disclosure can be administered by oraladministration.

When a pharmaceutical composition comprising a compound of the presentdisclosure is administered, dosages are expressed based on the amount ofactive pharmaceutical ingredient, i.e., the amount of nitroxyl donorcompound(s) of the disclosure present in the pharmaceutical composition.

For intravenous administration, the dose can usefully be expressed perunit time, either as a fixed amount per unit time or as a weight-basedamount per unit time.

In various embodiments, a compound or pharmaceutical composition of thedisclosure is administered intravenously in an amount of at least about0.1 μg/kg/min, at least about 0.2 μg/kg/min, at least about 0.3μg/kg/min, at least about 0.4 μg/kg/min, at least about 0.5 μg/kg/min,at least about 1 μg/kg/min, at least about 2.5 μg/kg/min, at least about5 μg/kg/min, at least about 7.5 μg/kg/min, at least about 10 μg/kg/min,at least about 11 μg/kg/min, at least about 12 μg/kg/min, at least about13 μg/kg/min, at least about 14 μg/kg/min, at least about 15 μg/kg/min,at least about 16 μg/kg/min, at least about 17 μg/kg/min, at least about18 μg/kg/min, at least about 19 μg/kg/min, at least about 20 μg/kg/min,at least about 21 μg/kg/min, at least about 22 μg/kg/min, at least about23 μg/kg/min, at least about 24 μg/kg/min, at least about 25 μg/kg/min,at least about 26 μg/kg/min, at least about 27 μg/kg/min, at least about28 μg/kg/min, at least about 29 μg/kg/min, at least about 30 μg/kg/min,at least about 31 μg/kg/min, at least about 32 μg/kg/min, at least about33 μg/kg/min, at least about 34 μg/kg/min, at least about 35 μg/kg/min,at least about 36 μg/kg/min, at least about 37 μg/kg/min, at least about38 μg/kg/min, at least about 39 μg/kg/min, or at least about 40μg/kg/min.

In various embodiments, the compound or pharmaceutical composition ofthe present disclosure is administered intravenously in an amount of nomore than about 100 μg/kg/min, no more than about 90 μg/kg/min, no morethan about 80 μg/kg/min, no more than about 70 μg/kg/min, no more thanabout 60 μg/kg/min, no more than about 50 μg/kg/min, no more than about49 μg/kg/min, no more than about 48 μg/kg/min, no more than about 47μg/kg/min, no more than about 46 μg/kg/min, no more than about 45μg/kg/min, no more than about 44 μg/kg/min, no more than about 43μg/kg/min, no more than about 42 μg/kg/min, no more than about 41μg/kg/min, no more than about 40 μg/kg/min, no more than about 39μg/kg/min, no more than about 38 μg/kg/min, no more than about 37μg/kg/min, no more than about 36 μg/kg/min, no more than about 35μg/kg/min, no more than about 34 μg/kg/min, no more than about 33μg/kg/min, no more than about 32 μg/kg/min, no more than about 31μg/kg/min, or no more than about 30 μg/kg/min

In some embodiments, the compound or pharmaceutical composition of thepresent disclosure is administered intravenously in an amount rangingfrom about 0.1 μg/kg/min to about 100 μg/kg/min, about 1 μg/kg/min toabout 100 μg/kg/min, about 2.5 μg/kg/min to about 100 μg/kg/min, about 5μg/kg/min to about 100 μg/kg/min, about 10 μg/kg/min to about 100μg/kg/min, about 1.0 μg/kg/min to about 80 μg/kg/min, from about 10.0μg/kg/min to about 70 μg/kg/min, from about 20 μg/kg/min to about 60μg/kg/min, from about 15 μg/kg/min to about 50 μg/kg/min, from about0.01 μg/kg/min to about 1.0 μg/kg/min, from about 0.01 μg/kg/min toabout 10 μg/kg/min, from about 0.1 μg/kg/min to about 1.0 μg/kg/min,from about 0.1 μg/kg/min to about 10 μg/kg/min, from about 1.0 μg/kg/minto about 5 μg/kg/min, from about 70 μg/kg/min to about 100 μg/kg/min, orfrom about 80 μg/kg/min to about 90 μg/kg/min.

In particular embodiments, the compound or pharmaceutical composition ofthe present disclosure is administered intravenously in an amountranging from about 10 μg/kg/min to about 50 μg/kg/min, about 20μg/kg/min to about 40 μg/kg/min, about 25 μg/kg/min to about 35μg/kg/min, or about 30 μg/kg/min to about 40 μg/kg/min. In particularembodiments, a compound or pharmaceutical composition of the presentdisclosure is administered intravenously in an amount of from about 20μg/kg/min to about 30 μg/kg/min.

In a variety of embodiments, including various oral administrationembodiments, the compounds or pharmaceutical compositions of thedisclosure are administered according to a weight-based daily dosingregimen, either as a single daily dose (QD) or in multiple divided dosesadministered, e.g., twice a day (BID), three times a day (TID), or fourtimes a day (QID).

In certain embodiments, the nitroxyl donating compound or pharmaceuticalcomposition of the disclosure is administered in a dose of at leastabout 0.5 mg/kg/d, at least about 0.75 mg/kg/d, at least about 1.0mg/kg/d, at least about 1.5 mg/kg/d, at least about 2 mg/kg/d, at leastabout 2.5 mg/kg/d, at least about 3 mg/kg/d, at least about 4 mg/kg/d,at least about 5 mg/kg/d, at least about 7.5 mg/kg/d, at least about 10mg/kg/d, at least about 12.5 mg/kg/d, at least about 15 mg/kg/d, atleast about 17.5 mg/kg/d, at least about 20 mg/kg/d, at least about 25mg/kg/d, at least about 30 mg/kg/d, at least about 35 mg/kg/d, at leastabout 40 mg/kg/d, at least about 45 mg/kg/d, at least about 50 mg/kg/d,at least about 60 mg/kg/d, at least about 70 mg/kg/d, at least about 80mg/kg/d, at least about 90 mg/kg/d, or at least about 100 mg/kg/d.

In certain embodiments, the nitroxyl donating compound or pharmaceuticalcomposition of the disclosure is administered at a dose of no more thanabout 100 mg/kg/d, no more than about 100 mg/kg/d, no more than about 90mg/kg/d, no more than about 80 mg/kg/d, no more than about 80 mg/kg/d,no more than about 75 mg/kg/d, no more than about 70 mg/kg/d, no morethan about 60 mg/kg/d, no more than about 50 mg/kg/d, no more than about45 mg/kg/d, no more than about 40 mg/kg/d, no more than about 35mg/kg/d, no more than about 30 mg/kg/d.

In a variety of embodiments, the dose is from about 0.001 mg/kg/d toabout 10,000 mg/kg/d. In certain embodiments, the dose is from about0.01 mg/kg/d to about 1,000 mg/kg/d. In certain embodiments, the dose isfrom about 0.01 mg/kg/d to about 100 mg/kg/d. In certain embodiments,the dose is from about 0.01 mg/kg/d to about 10 mg/kg/d. In certainembodiments, the dose is from about 0.1 mg/kg/d to about 1 mg/kg/d. Incertain embodiments, the dose is less than about 1 g/kg/d.

In certain embodiments, a compound or pharmaceutical composition of thedisclosure is administered in a dose range in which the low end of therange is any amount from about 0.1 mg/kg/day to about 90 mg/kg/day andthe high end of the range is any amount from about 1 mg/kg/day to about100 mg/kg/day (e.g., from about 0.5 mg/kg/day to about 2 mg/kg/day inone series of embodiments and from about 5 mg/kg/day to about 20mg/kg/day in another series of embodiment).

In particular embodiments, the compound or pharmaceutical composition ofthe disclosure is administered in a dose range of about 3 to about 30mg/kg, administered from once a day (QD) to three times a day (TID).

In certain embodiments, compounds or pharmaceutical compositions of thedisclosure are administered according to a flat (i.e., non-weight-based)dosing regimen, either as a single daily dose (QD) or in multipledivided doses administered, e.g., twice a day (BID), three times a day(TID), or four times a day (QID).

In various embodiments, the compound or pharmaceutical composition ofthe disclosure is administered at a dose of at least about 0.01grams/day (g/d), at least about 0.05 g/d, at least about 0.1 g/d, atleast about 0.5 g/d, at least about 1 g/d, at least about 1.5 g/d, atleast about 2.0 g/d, at least about 2.5 g/d, at least about 3.0 g/d, orat least about 3.5 g/d.

In various embodiments, the compound or pharmaceutical composition ofthe disclosure is administered at a dose of no more than about 5 g/d, nomore than about 4.5 g/d, no more than about 4 g/d, no more than about3.5 g/d, no more than about 3 g/d, no more than about 2.5 g/d, or nomore than about 2 g/d.

In certain embodiments, the compound or pharmaceutical composition ofthe disclosure is administered in a dose of about 0.01 grams per day toabout 4.0 grams per day. In certain embodiments, a compound orpharmaceutical composition of the disclosure can be administered at adose in which the low end of the range is any amount from about 0.1mg/day to about 400 mg/day and the high end of the range is any amountfrom about 1 mg/day to about 4000 mg/day. In certain embodiments, thecompound or pharmaceutical composition is administered in a dose ofabout 5 mg/day to about 100 mg/day. In various embodiments, the compoundor pharmaceutical composition is administered at a dose of from about150 mg/day to about 500 mg/day.

The dosing interval for parenteral or oral administration can beadjusted according to the needs of the patient. For longer intervalsbetween administrations, extended release or depot formulations can beused.

A compound or pharmaceutical composition as disclosed herein can beadministered prior to, at substantially the same time with, or afteradministration of an additional therapeutic agent. The administrationregimen can include pretreatment and/or co-administration with theadditional therapeutic agent. In such case, the compound orpharmaceutical composition and the additional therapeutic agent can beadministered simultaneously, separately, or sequentially.

Examples of administration regimens include without limitation:administration of each compound, pharmaceutical composition ortherapeutic agent in a sequential manner; and co-administration of eachcompound, pharmaceutical composition or therapeutic agent in asubstantially simultaneous manner (e.g., as in a single unit dosageform) or in multiple, separate unit dosage forms for each compound,pharmaceutical composition or therapeutic agent.

It will be appreciated by those in the art that the “effective amount”or “dose” (“dose level”) will depend on various factors such as theparticular administration mode, administration regimen, compound, andpharmaceutical composition selected, as well as the particular conditionand patient being treated. For example, the appropriate dose level canvary depending upon the activity, rate of excretion and potential fortoxicity of the specific compound or pharmaceutical compositionemployed; the age, body weight, general health, gender and diet of thepatient being treated; the frequency of administration; the othertherapeutic agent(s) being co-administered; and the type and severity ofthe condition.

4.7 Kits Comprising the Compounds or Pharmaceutical Compositions

The disclosure provides kits comprising a compound or a pharmaceuticalcomposition disclosed herein. In a particular embodiment, the kitcomprises a compound or a pharmaceutical composition disclosed herein,each in dry form, and a pharmaceutically acceptable liquid diluent.

In particular embodiments, either a compound in dry form or apharmaceutical composition in dry form contains about 2.0% or less waterby weight, about 1.5% or less water by weight, about 1.0% or less waterby weight, about 0.5% or less water by weight, about 0.3% or less waterby weight, about 0.2% or less water by weight, about 0.1% or less waterby weight, about 0.05% or less water by weight, about 0.03% or lesswater by weight, or about 0.01% or less water by weight.

Pharmaceutically acceptable liquid diluents are known in the art andinclude but are not limited to sterile water, saline solutions, aqueousdextrose, glycerol, glycerol solutions, and the like. Other examples ofsuitable liquid diluents are disclosed by Nairn, “Solutions, Emulsions,Suspensions and Extracts,” pp. 721-752 in Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins,Baltimore, Md., 2000).

In one embodiment, the kit further comprises instructions for using thecompound or pharmaceutical composition. The instructions can be in anyappropriate form, such as written or electronic form. In anotherembodiment, the instructions can be written instructions. In anotherembodiment, the instructions are contained in an electronic storagemedium (e.g., magnetic diskette or optical disk). In another embodiment,the instructions include information as to the compound orpharmaceutical composition and the manner of administering the compoundor pharmaceutical composition to a patient. In another embodiment, theinstructions relate to a method of use disclosed herein (e.g., treating,preventing and/or delaying onset and/or development of a conditionselected from cardiovascular diseases, ischemia/reperfusion injury,pulmonary hypertension and other conditions responsive to nitroxyltherapy).

In another embodiment, the kit further comprises suitable packaging.Where the kit comprises more than one compound or pharmaceuticalcomposition, the compounds or pharmaceutical compositions can bepackaged patiently in separate containers, or combined in one containerwhen cross-reactivity and shelf life permit.

5. EXAMPLES

The following examples are presented for illustrative purposes andshould not serve to limit the scope of the disclosed subject matter.

5.1 Synthesis of Compounds

The compounds disclosed herein can be made according to the methodsdisclosed below or by procedures known in the art. Starting materialsfor the reactions can be commercially available or can be prepared byknown procedures or obvious modifications thereof. For example, some ofthe starting materials are available from commercial suppliers such asSigma-Aldrich (St. Louis, Mo.). Others can be prepared by procedures orobvious modifications thereof disclosed in standard reference texts suchas March's Advanced Organic Chemistry (John Wiley and Sons) and Larock'sComprehensive Organic Transformations (VCH Publishers).

Example 1: Preparation of N-Hydroxy-5-methylfuran-2-sulfonamide (1)

To a solution of hydroxylamine (0.92 mL of a 50% aqueous solution; 13.8mmol) in THF (6 mL) and water (2 mL) cooled to 0° C. was added5-methylfuran-2-sulfonyl chloride (1 g, 5.5 mmol) as a solution in THF(6 mL) dropwise so as to maintain the temperature below 10° C. Thereaction was stirred for 5 minutes, after which time TLC (1:1hexane:ethyl acetate (H:EA)) showed substantially complete consumptionof the sulfonyl chloride. The reaction was diluted twice with 50 mLdichloromethane (DCM) and the organic portion was separated and washedwith water (10 mL). The organic portion was dried over sodium sulfate,filtered and concentrated under reduced pressure. The product waschromatographed by silica gel chromatography eluting with heptanes:EtOAcfollowed by trituration with heptane to provide the title compound as ayellow solid (0.59 g, yield 61%). LC-MS t_(R)=0.91 min; ¹H NMR (DMSO,500 MHz) δ ppm 9.82 (1H, d, J=3.1 Hz), 9.64 (1H, d, J=3.2 Hz), 7.10 (1H,d, J=3.4 Hz), 6.36 (1H, d, J=3.4 Hz), 2.36 (3H, s).

Example 2: Preparation ofN-Hydroxy-3-methanesulfonylbenzene-1-sulfonamide (2)3-Methanesulfonylbenzene-1-sulfonyl chloride

The intermediate 3-methanesulfonylbenzene-1-sulfonyl chloride wassynthesized according to the methods disclosed in Park et al., J. Med.Chem. 51(21):6902-6915 (2008). Specifically, methyl sulfonyl benzene(110 g, 0.7 mol) was heated for 18 hours at 90° C. in chlorosulfonicacid (450 mL, 6.7 mol) after which time the reaction mixture was allowedto cool to a temperature of about 21° C. before slowly being poured ontocrushed ice. The resulting slurry was twice extracted into EtOAc (2 Lfor each extraction). The organic portions were combined and washed withbrine (50 mL) before being dried over sodium sulfate, filtered andconcentrated under reduced pressure to provide the intermediate sulfonylchloride as an off white solid (125 g, yield 75%). ¹H NMR (400 MHz,CDCl₃) δ ppm 8.61 (1 h, t, J=1.7 Hz), 8.35-8.31 (2H, m), 7.90 (1H, t,J=7.9 Hz), 3.15 (3H, s).

N-Hydroxy-3-methanesulfonylbenzene-1-sulfonamide

To a solution of aqueous hydroxylamine (16 mL of a 50% aqueous solution,245 mmol) in THF (150 mL) and water (25 mL) cooled to −5° C. was slowlyadded 3-methanesulfonylbenzene-1-sulfonyl chloride (25 g, 98 mmol) whilemaintaining a reaction temperature of less than 10° C. The reaction wasmaintained at this temperature until substantially complete consumptionof the sulfonyl chloride was observed (about 5 min), after which timethe reaction was diluted with DCM (250 mL), the organic portion wasseparated and washed twice with 50 mL of water. The aqueous extractswere combined and rewashed twice with DCM (250 mL for each wash). All ofthe organic portions were combined, dried over sodium sulfate, filteredand concentrated under reduced pressure to provide the title compound asa beige solid. Trituration was carried out using heptanes:EtOAc (1:1;v:v) to provide the title compound as a beige solid (14 g, yield 56%).LC-MS t_(R)=0.90 min; High Resolution Mass Spectroscopy (HRMS):theoretical (C₇H₉NO₅S₂)=249.9844, measured=249.9833; ¹H NMR (500 MHz,DMSO-d₆) δ ppm 9.85 (2H, q, J=3.3 Hz), 8.31 (1H, t, J=1.6 Hz), 8.28 (1H,dt, J=7.8, 1.3 Hz), 8.14-8.19 (1H, m), 7.93 (1H, t, J=7.9 Hz), 3.32 (3H,s).

5.2 Example 3: Nitroxyl Production as Determined Via N₂O Quantification

Nitrous oxide (N₂O) is produced via the dimerization and dehydration ofHNO, and is the most common marker for nitroxyl production (Fukuto etal., Chem. Res. Toxicol. 18:790-801 (2005)). Nitroxyl, however, can alsobe partially quenched by oxygen to provide a product that does notproduce N₂O (see Mincione et al., J. Enzyme Inhibition 13:267-284(1998); and Scozzafava et al., J. Med. Chem. 43:3677-3687 (2000)). Usingeither nitrous oxide gas or Angeli's salt (AS) as a standard, therelative amounts of N₂O released from compounds of the disclosure wasexamined via gas chromatography (GC) headspace analysis.

A procedure for determining the relative amounts of N₂O released fromcompounds of the disclosure is as follows. GC was performed on anAgilent gas chromatograph equipped with a split injector (10:1splitting), microelectron capture detector, and a HP-MOLSIV 30 m×0.32mm×25 μm molecular sieve capillary column. Helium was used as thecarrier (4 mL/min) gas and nitrogen was used as the make-up (20 mL/min)gas. The injector oven and the detector oven were kept at 200° C. and325° C., respectively. All nitrous oxide analyses were performed withthe column oven held at a constant temperature of 200° C.

All gas injections were made using an automated headspace analyzer. Vialpressurization was 15 psi. The analyzer's sample oven, sampling valve,and transfer line were kept at 40° C., 45° C., and 50° C., respectively.The oven stabilization, vial pressurization, loop fill, loopequilibration, and sample injection times were 1.00 min., 0.20 min.,0.20 min., 0.05 min., and 1.00 min., respectively.

All determinations used a batch of nominal 20 mL headspace vials withvolumes pre-measured for sample uniformity (actual vial volume varied by<2.0% relative standard deviation (n=6)). The average vial volume forthe batch was determined from six randomly-selected vials by calculatingthe weight difference between the capped and sealed empty (i.e.,air-filled) vial and the capped and sealed deionized water-filled vialusing the known density of deionized water, then averaging. Blanks wereprepared by sealing and capping two vials then purging each for 20seconds with a gentle argon stream. Nitroxyl standards were prepared bysealing and capping four vials then purging each for 1 minute with agentle stream, from a gas cylinder, of a 3000 ppm nitroxyl standard.

CXL-1020 (N-hydroxy-2-methanesulfonylbenzene-1-sulfonamide) “standards”were prepared by, in duplicate, accurately weighing 10±0.5 mg ofCXL-1020 and adding it to each 4 mL vial. Using an auto pipette, 1 mL ofargon-purged anhydrous DMF (Sigma-Aldrich) was added to each 4 mL vialto form a CXL-1020 stock solution for each sample and the vials werecapped and shaken and/or sonicated to insure complete dissolution uponvisual observation. Using an auto pipette, 20 mL vials were charged with5 mL of PBS (purged for at least 30 min. with argon prior to use),purged with argon for at least 20 sec., and sealed with a rubber septum.Using a 50 μL syringe, 50 μL of the CXL-1020 stock solution was injectedinto each 20 mL vial containing the PBS.

Samples were prepared as follows. In duplicate, 18±1 mg of each samplewas accurately weighed into each 4 mL vial. Using an auto pipette, 1 mLof argon-purged anhydrous DMF was added to each 4 mL vial to form asample stock solution for each sample and the vials were capped andshaken and/or sonicated to insure complete sample dissolution uponvisual observation. Using an auto pipette, 20 mL vials were charged with5 mL of PBS (purged for at least 30 min. with argon prior to use),purged with argon for at least 20 sec., and sealed with a rubber septum.The vials were equilibrated for at least 10 min. at 37° C. in a dryblock heater. Thereafter, using a 50 μL syringe, 50 μL of a sample stocksolution was injected into each 20 mL vial containing the PBS. The vialswere then held at 37° C. in the dry block heater for a time period suchthat the sum of the time spent in the dry block heater plus the timespent in the automated headspace analyzer oven before sample injectionequaled the desired incubation time.

The sequence for auto-injection was as follows: blank replicate 1, blankreplicate 2, N₂O standard replicate 1, N₂O standard replicate 2,CXL-1020 standard replicate 1, CXL-1020 standard replicate 2, sample 1replicate 1, sample 1 replicate 2, sample 2 replicate 1, sample 2replicate 2, etc., concluding with N₂O standard replicate 3, and N₂Ostandard replicate 4. An EXCEL spreadsheet is used for inputting datathus determined and calculating, for each sample, the relative N₂O yieldin percent for each incubation time. The results obtained are providedin Table 2.

TABLE 2 Results of N₂O Headspace Analysis Relative N₂O Yield RelativeN₂O Yield (360 minute Compound (90 minute incubation) incubation)N-Hydroxy-5-methylfuran- 52% N/A 2-sulfonamide (1) N-Hydroxy-3- 82% 94%methanesulfonylbenzene- 1-sulfonamide (2)

For compounds of formulas (3) and (4), determinations are as describedabove except enzyme activated samples are also prepared as follows: (i)accurately weigh 50 mg of porcine liver esterase (PLE, E3019-20KU,crude, Sigma-Aldrich) into a 20 mL headspace vial; (ii) using an autopipette, 5 mL of argon-purged anhydrous PBS is added to form a PLE stocksolution; (iii) the vial is capped and shaken to insure completedissolution upon visual observation; (iv) samples of nitroxyl donors areprepared as disclosed above except 4.75 mL of PBS is added instead of 5mL; and (v) using an auto pipette, the 20 mL vials are then charged with250 μmL of PLE stock solution prior to sample addition. The sequence forauto-injection is as follows: blank replicate 1, blank replicate 2, N₂Ostandard replicate 1, N₂O standard replicate 2, CXL-1020 standardreplicate 1, CXL-1020 standard replicate 2, sample 1 (no PLE) replicate1, sample 1 (no PLE) replicate 2, sample 1 (with PLE) replicate 1,sample 1 (with PLE) replicate 2, sample 2 (no PLE) replicate 1, sample 2(no PLE) replicate 2, sample 2 (with PLE) replicate 1, sample 2 (withPLE) replicate 2, etc., concluding with N₂O standard replicate 3, andN₂O standard replicate 4.

Another procedure for determining the relative amounts of N₂O releasedfrom compounds of the disclosure is as follows. GC is performed on aVarian CP-3800 instrument equipped with a 1041 manual injector, electroncapture detector, and a 25 m 5 Å molecular sieve capillary column. Grade5.0 nitrogen is used as both the carrier (8 mL/min) and the make-up (22mL/min) gas. The injector oven and the detector oven are kept at 200° C.and 300° C., respectively. All nitrous oxide analyses are performed withthe column oven held at a constant temperature of 150° C. All gasinjections are made using a 100 μL gas-tight syringe with a sample-lock.Samples are prepared in 15 mL amber headspace vials with volumespre-measured for sample uniformity (actual vial volume ranges from 15.19to 15.20 mL). Vials are charged with 5 mL of PBS containingdiethylenetriamine pentaacetic anhydride (DTPA), purged with argon, andsealed with a rubber septum. The vials are equilibrated for at least 10minutes at 37° C. in a dry block heater. A 10 mM stock solution of AS isprepared in 10 mM sodium hydroxide, and solutions of the nitroxyl donorsare prepared in either acetonitrile or methanol and used immediatelyafter preparation. From these stock solutions, 50 μL is introduced intoindividual thermally-equilibrated headspace vials using a 100 μLgas-tight syringe with a sample-lock to provide final substrateconcentrations of 0.1 mM. Substrates are then incubated for 90 minutesor 360 minutes. The headspace (60 μL) is then sampled and injected fivesuccessive times into the GC apparatus using the gas-tight syringe witha sample lock. This procedure is repeated for two or more vials perdonor.

5.3. Example 4: In Vitro Stability of Nitroxyl Donors in Plasma

Compound (1), compound (2), and CXL-1020 were tested for their stabilityin plasma. The assay system comprised (i) PBS, or plasma from rat, dogor human (at least 3 donors, male, pooled) at pH 7.4, and (ii) for testsconducted in plasma, an anticoagulant (sodium heparin or sodiumcitrate). Each test compound (5 μM) was incubated in PBS or plasma at37° C. on a THERMOMIXER® with shaking. Three samples (n=3) were taken ateach of seven sampling time points: 0, 10, 30, 60, 90, 180 and 360minutes. The samples were immediately combined with 3 volumes (i.e., 3times the volume of PBS or plasma) of acetonitrile containing 1% formicacid and an internal standard to terminate the reaction. AB SCIEX API3000 LC-MS/MS analysis of the test compounds was performed without astandard curve. Halflives (T_(1/2)) of the test compounds weredetermined from graphs of the percent remaining values using the peakarea response ratio. The half-lives determined are provided in Table 3.

TABLE 3 Half-lives (T_(1/2)) of Nitroxyl Donors T_(1/2) T_(1/2) T_(1/2)T_(1/2) (minutes) (minutes) (minutes) (minutes) Compound PBS Rat DogHuman CXL-1020 2 N/A N/A 2 N-Hydroxy-5- 68 40 25 65 methylfuran-2-sulfonamide (1) N-Hydroxy-3- 50 20 33 37 methanesulfonylbenzene-1-sulfonamide (2)

For measuring half-lives of compounds of formula (3) or formula (4), astock solution of pig liver esterase (PLE) is added to the PBS or plasmaprior to addition of said compound.

5.4. Example 5: Solubility of Nitroxyl Donors

Initially, the solubilities of the compounds of formula (1) and formula(2) were measured by visual assessment at 100 μg/mL and 1000 μg/mL in apH 4 buffer. The buffer was prepared by mixing 660 mL of Solution A(10.5023 g of citric acid dissolved in 1 L of water) and 450 mL ofSolution B (14.7010 g of sodium citrate tribasic dihydrate dissolved in1 L of water). The pH of the buffer was 3.98 as measured by pH meter.

Each compound was shaken for about 5 minutes in the pH 4 buffer solutionprepared above at two concentration points (100 μg/mL and 1000 μg/mL)and the solubility was observed visually. The results obtained arepresented in Table 4.

TABLE 4 Solubility in pH 4 Buffer at 100 μg/mL and 1000 μ/mL SolubilityCompound Solubility (100 μg/mL) (1000 μg/mL) N-Hydroxy-5- Y Ymethylfuran-2- sulfonamide (1) N-Hydroxy-3 Y Y methanesulfonylbenzene-1-sulfonamide (2) N = Not soluble after shaking for 5 minutes in pH = 4buffer Y = Soluble after shaking for 5 minutes in pH = 4 buffer

Additionally, a sample of the compound of formula (1) was prepared inwater to determine the approximate solubility of the compound in theabsence of excipients (e.g., CAPTISOL®). A concentration ofapproximately 300 mg/mL was achieved, not accounting for the volumecontribution of the compound. The pH of the sample was determined to be2.8, which was adjusted to the target of 4.0 using 0.1 N NaOH. Upon pHadjustment, precipitation of a small amount of solid was observed. Theclear solution was diluted in acetonitrile and analyzed by HPLC,resulting in an observed solution concentration of 268 mg/mL. A similaranalysis was performed for the compound of formula (2). The compound offormula (2) has a solubility of approximately 10 mg/mL.

5.5 Example 6: Hemodynamic Efficacy of Nitroxyl Donors in Normal andHeart Failure Canines (Tachycardia-Pacing Model) 5.5.1 Materials andMethods

The cardiovascular effects of nitroxyl donors were examined by means ofpressure-volume (PV) curve (loops) analysis in conscious,sling-restrained beagle dogs. Animals were allowed free access todrinking water and a commercial canine diet under standard laboratoryconditions. Fluorescent lighting was provided via an automatic timer forapproximately 12 hours per day. On occasion, the dark cycle wasinterrupted intermittently due to study-related activities. Temperatureand humidity were monitored and recorded daily and maintained to themaximum extent possible between 64° F. and 84° F. and 30% to 70%,respectively. The dogs were acclimated for a period of at least 1 weekprior to surgery. Following surgery and recovery the animals wereacclimated to sling restraint for a period up to 4.5 hours. Animals werefasted overnight prior to surgery.

Surgical Procedure

Anesthesia

An indwelling venous catheter was placed in a peripheral vein (e.g.,cephalic) for administration of anesthetic. General anesthesia wasinduced intravenously (bolus) with buprenorphine (about 0.015 mg/kg)followed by an intravenous bolus of propofol (about 6 mg/kg).Additionally, a prophylactic antibiotic (cefazolin 20 to 50 mg/kg viai.v.) was given upon induction. A cuffed tracheal tube was placed andused to ventilate mechanically ventilate the lungs with 100% O₂ via avolume-cycled animal ventilator (about 12 breaths/minute with a tidalvolume of about 12.5 mL/kg) in order to sustain PaCO₂ values within thephysiological range. Anesthesia was maintained with inhaled isoflurane(1% to 3%).

Cardiovascular Instrumentation

Once a stable (surgical) plane of anesthesia had been established, aleft-thoracotomy was performed (under strict aseptic conditions) andeach animal was chronically instrumented with sono-micrometry crystalsproviding left-ventricular (LV) dimensions/volume. Additionally, afluid-filled catheter and a solid-state monometer were placed in theleft ventricle for pressure monitoring. A fluid-filled catheter wasplaced in the right ventricle (RV) and the aorta (Ao) for pressuremonitoring/test article administration. A hydraulic (In-Vivo Metrics)occluder was placed/secured around the inferior vena cava (IVC), inorder to allow its controlled constriction for the generation of LVpressure-volume curves during heterometric auto-regulation. Thecatheters/wires were aseptically tunneled and externalized between thescapulae. Over the course of the study, fluid-filled catheters wereregularly (at least once weekly) flushed with a locking-solution inorder to prevent both clotting and bacterial growth (2-3 mL ofTaurolidine-Citrate solution, TCS-04; Access Technologies).

Pacemaker Implantation

Following the cardiovascular instrumentation, the right jugular vein wascarefully exposed and cannulated with a bipolar pacing lead/catheter(CAPSUREFIX® Novus; Medtronic). Under fluoroscopic guidance, this pacinglead was advanced normograde into the right ventricle and activelyaffixed (screwed in) to the apical endocardium. The proximal end of thelead was secured to the pacing device (Kappa 900; Medtronic).Subsequently, the pacemaker was placed/secured in a subcutaneous pocketin the neck.

Considering that the heart was exposed via a thoracotomy, a bipolarpacing wire was secured in the right ventricular mid-myocardium. Thispacing lead was tunneled/externalized between the scapulae, and used inconjunction with an external impulse generator/pacemaker. The implantedendocardial pacemaker was used as a back-up to the external/epicardialpacemaker.

Recovery

Prior to closure of the chest from the thoracotomy, a chest tube wasplaced for drainage of any fluid and/or gas that accumulated from thesurgical procedure. The tube was aspirated twice daily until the amountof fluid removed was less than 35 mL per aspiration in an approximately24 hour period. The chest tube was then removed.

All animals were administered a prophylactic antibiotic (cefazolin 20 to50 mg/kg via i.v.) and pain medication (meloxicam at about 0.2 mg/kg viai.v.). If necessary, an additional analgesic was also administered whichincluded a fentanyl patch (25 to 50 mcg/hour). All surgical incisionswere closed in layers; the underlying musculature was closed withabsorbable sutures and the skin was closed with staples.

Following surgery, the animals were allowed to recover for at least 14days. Cephalexin (20 to 50 mg/kg) was administered orally BID for atleast 7 days and meloxicam (0.1 mg/kg) was administered SID orally orsubcutaneously for at least 2 days after surgery. Throughout therecovery phase, the animals were observed daily for routine signs ofrecovery and the wound sites were observed for any signs of potentialinfections. Animals experiencing pain, distress and/or infections werebrought to the attention of the attending veterinarian and the studydirector. The skin incision staples were not removed for at least 7 daysafter surgery.

Induction of Heart Failure

Following a recovery from surgery and/or sufficient washout period fromdosing with a nitroxyl donor, animals were subjected to a 3-weekoverdrive pacing (210 ppm) protocol aimed to trigger left-ventriculardysfunction/remodeling consistent with the heart failure syndrome. Inshort, via the implanted pacemaker/right-ventricular lead, theventricle(s) was asynchronously and continuously paced at 210 beats perminute (bpm). Left-ventricular remodeling (and heart failure induction)were confirmed by both echocardiographic (e.g., ejection fraction (EF))decrease from about 60% to a target of about 35%, left ventricular (LV)dilatation) and neuro-humoral (e.g., N-terminal pro-brain natriureticpeptide (NT proBNP) elevation to greater than 1800 pM/L from a baselineof about 300 pM/L) changes after approximately 3 weeks of pacing.Echocardiographs and blood samples were collected in the absence ofpacing (for at least 15 min).

5.5.2 Results

Hemodynamic Efficacy Assessments

The animals (normal or heart failure) were studied during treatment withboth vehicle (control) and a nitroxyl donor (either CXL-1020, a compoundof formula (1) or a compound of formula (2)). At each dosing period,conscious sling-restrained animals were continuously monitored for up totwo to three hours. Following hemodynamic stabilization, infusion of thevehicle was started. Shortly thereafter, left-ventricular pre-load wasacutely reduced by means of brief vena cava occlusions (via transientinflation of the vessel occluder) in order to generate a family ofpressure-volume curves/loops; up to three occlusions were performed,allowing for hemodynamic recovery between tests. Infusion of the vehiclewas continued and after 30 min another (baseline) set of hemodynamicdata was collected. Following collection of baseline hemodynamic data,infusion of the nitroxyl donor compound being tested was initiated andderived hemodynamic/functional parameters were obtained/performed at upto four (4) time points selected from the following: at 30, 60, 90, 120,and 180 minutes after the onset of vehicle/test compound infusion. Forthe placebo or time-control treatment group, each animal wasadministered an infusion of an appropriate placebo for up to 180minutes. In all cases, the test compound was delivered at a constantintravenous infusion rate of 1 mL/kg/hr and was compared at a molarequivalent dose rate.

The resulting left-ventricular pressure and volume data were analyzed inorder to generate relationships representing the contractile andenergetic state of the myocardium. Systolic arterial pressure (SAP),diastolic arterial pressure (DAP), and mean arterial pressure (MAP) werecollected. Left-ventricular mechanical and/or geometrical indices wereobtained from the pressure (ESP, EDP, dP/dt max/min, time-constant ofrelaxation-tau [based on mono-exponential decay with non-zeroasymptote]) and volume (end-systolic volume (ESV), end diastolic volume(EDV), stroke volume (SV)) signal. In addition, the followingmeasurements were derived from left-ventricular pressure-volume data (PVloops) generated during brief periods of preload reduction: pressurevolume area (PVA) and stroke work (SW), end-systolic (ESPVR) andend-diastolic (EDPVR) pressure volume relationships, and end systolicpressure and stroke volume relationship (arterial elastance (Ea)).Representative data obtained from studies in normal dogs and heartfailure dogs is shown in Table 5 and Table 6, respectively. A SVR(systemic vascular resistance) decrease correlates with vasodilation.

TABLE 5 Hemodynamic Parameters for Nitroxyl Donors in Normal Canines (%Change from Baseline) Compound Control CXL-1020 (1) (2) Dose Rate 0 10050 100 (μmol/kg/ min) Number of 3  6  8  4 Animals HR −2.21 ± 1.51  6.71± 4.72 −4 ± 2  −6.17 ± 5.58 ESP  −1.8 ± 0.58 −17.79 ± 3.09 −18 ± 2 −15.22 ± 2.39 EDV  2.62 ± 0.42 −20.51 ± 7.63 −6 ± 2 −17.41 ± 1.58 Tau11.14 ± 1.15  −6.58 ± 4.53 −6 ± 1  −6.40 ± 7.11 SW −2.80 ± 1.26 −13.96 ±5.51 −11 ± 4  −17.56 ± 2.66 ESPVR −3.20 ± 1.15  28.25 ± 8.69 19 ± 1 25.87 ± 5.04 PRSW −0.78 ± 0.38  12.60 ± 2.96 12 ± 1  12.88 ± 1.12Abbreviations: HR: Heart rate. Increased HR, either due to reflexresponse to low blood pressure or due to a primary drug effect on theheart, is bad. ESP: End systolic pressure - similar to MAP below. EDP orLVEDP: End diastolic pressure (left ventricular). Correlates withpulmonary pressures. A decrease indicates a reduction of pulmonarycongestion (a key objective of acute heart failure therapy). Tau: Anindex of lusitropy, or relaxation of the heart during diastole. Decreaseis positive and indicates improved diastolic performance. SW: Strokework. Measure of how much work the heart exerts to create a given amountof forward flow. ESPVR: End systolic pressure volume relationship. Ameasure of inotropy/contractility (a key objective of acute heartfailure therapy). Increases indicate improved cardiac performance andefficiency. PRSW: Preload recruitable stroke work - similar to ESPVRabove. SV: Stroke volume. The amount of blood ejected from the leftventricle with each beat of the heart. An inotrope should increase this,given identical loading conditions. MAP OR MBP: Mean arterial pressureor mean blood pressure. Small drops are positive and evidence ofvasodilation. EDV or LVEDV: End diastolic volume (left ventricular).Index of the degree of filling in diastole. A decrease indicates areduction in volume overload.

TABLE 6 Hemodynamic Parameters for Nitroxyl Donors in Heart FailureCanines (% Change from Baseline) Compound Control CXL-1020 (1) (2) Dose0 100 75 100 Rate (μmol/ kg/min) Number 3  6  6  4 of Animals HR −5.08 ±5.83   −0.23 ± 2.25 −6 ± 2 −1.36 ± 2.06 ESP 3.89 ± 2.11 −14.78 ± 3.24−17 ± 1  −13.83 ± 3.30  EDV 0.86 ± 0.86 −12.03 ± 3.72 −9 ± 2 −3.26 ±1.05 Tau 4.05 ± 4.72 −17.27 ± 1.39 −16 ± 4  −12.51 ± 2.72  SW 1.83 ±1.87 −12.01 ± 4.24 −9 ± 2 −9.41 ± 2.84 ESPVR −3.14 ± 0.87   45.42 ±16.48 29 ± 1 22.84 ± 5.69 PRSW −0.88 ± 0.68   21.97 ± 3.79 22 ± 1 17.91± 1.47 Abbreviations: HR, heart rate; ESP, end systolic pressure; EDV,end diastolic volume; Tau, time constant for relaxation; SW, strokework; ESPVR, end systolic pressure volume relationship; PRSW, preloadrecruitable stroke work.

FIG. 1 shows the hemodynamic profile 180 minutes after administration ofCXL-1020 and two compounds of the disclosure (compounds of formula (1)and formula (2)) using a tachycardia-pacing model of heart failure. Eachcompound was administered intravenously at a rate of 100 μg/kg/min. FIG.2 shows the hemodynamic profile of the compound of formula (1) atvarious dosages using a tachycardia-pacing model of heart failure. Theresults demonstrate that compounds of formula (1) and (2) havecomparable hemodynamic activity to CXL-1020 in both normal and failingcanine models. In particular, the compounds of formula (1) and (2)produce significant enhancement of inotropy and lusitropy, and modestreductions in blood pressure.

5.6 Example 7: Hemodynamic Efficacy of Nitroxyl Donors in Heart FailureCanines (Canine Microembolization Heart Failure Model) 5.6.1 Materialsand Methods

Heart failure was produced using healthy, conditioned, purpose-bredmongrel dogs (20-26 kg) using a sequential microembolization model.Coronary microembolization was performed until LV-ejection fraction(determined angiographically under anesthesia) was approximately 30% orlower. Two weeks were then provided after the last microembolization toensure stabilization of each animal prior to initiating experiments.

An initial dose-finding study (2-100 μg/kg/min for 40 min) was performedin 3 dogs to identify therapeutically relevant doses of a compound offormula (1). Based on these data, a primary group of six animals werestudied, receiving 3 or 10 μg/kg/min of a compound of formula (1) over a4 hour period, followed by one hour washout. Only one dose was studiedon a given day, the other at least one-week later, and the orderrandomized. Hemodynamic, ventriculographic, and echocardiographicmeasurements were made during left and right heart catheterizations inanesthetized dogs (induction: hydromorphone (0.22 mg/kg i.v.) anddiazepam (0.17 mg/kg i.v.), maintenance: 1-2% isofluorane).

LV end-systolic volume (ESV) and end-diastolic volumes (EDV) werecalculated from ventriculograms using the area-length method. Peakaortic blood velocity was obtained in the ascending aorta using flowDoppler for measurements of peak power index (PPI). LV fractional areaof shortening (FAS) was measured from LV short axis view at the level ofpapillary muscles obtained from 2-dimensional echocardiograms. Measuredindexes of LV diastolic function included deceleration time of mitralinflow velocity (DT), ratio of the integral of early mitral inflowvelocity (Ei) to velocity during atrial contraction (Ai) (Ei/Ai) and LVend-diastolic circumferential wall stress (EDWS).

Measurements of myocardial oxygen consumption were performed at baselineand at 4 hours after 10 μg/kg/min infusion. Specifically, arterial andcoronary sinus blood samples were simultaneously drawn at baseline andat the end of each study time point. Oxygen content was determined witha hemoximeter. Coronary artery blood velocity was measured with aDoppler flow velocity wire placed in the left circumflex coronary arteryproximal to the first marginal branch or in the left anterior descendingcoronary artery just proximal to the first diagonal branch. Blood flowwas estimated by calculating the cross-sectional area of the coronaryartery at the site of the velocity measurement from coronaryangiography. Total coronary blood flow was assumed to be twice the flowmeasured in the circumflex or left anterior descending coronary artery.Oxygen consumption of the left ventricle (MVO2) was calculated as theproduct of total coronary blood flow and the oxygen content differencebetween arterial and coronary sinus blood.

5.6.2 Results

Hemodynamic Efficacy Assessments

The animals were studied during treatment with both vehicle and anitroxyl donor. FIG. 3 shows the hemodynamic profile of the compound offormula (1) following induction of heart failure in dogs evaluated usinga canine microembolization heart failure model. The data is shown forfinal time point during infusion at two rates of infusion. The resultsdemonstrated that the compound of formula (1) had comparable hemodynamicactivity to CXL-1020.

5.7 Toxicological Studies with Nitroxyl Donors 5.7.1 Example 8: In VivoTrials with CXL-1020

During in vivo trials of the nitroxyl donor, CXL-1020(N-hydroxy-2-methanesulfonylbenzene-1-sulfonamide), a 14-day study wasconducted to evaluate tolerance in dogs treated with continuousinfusions of CXL-1020 at dose rates of up to 90 μg/kg/min. This firststudy found that CXL-1020 was tolerated when administered at a dose rateof 60 μg/kg/min. Unexpectedly, however, clinical pathology changesconsistent with an inflammation process, as reflected in changes inclinical pathology markers of inflammation, were observed at the 60μg/kg/min dose rate. To further investigate this undesirableside-effect, a follow-up 14-day study in dogs was initiated. Thefollow-up study needed to be terminated after only 4 days due to theappearance of other undesirable side-effects: the unexpected occurrenceof significant swelling and inflammation in the dogs' hind limbs whereinfusion catheters had been surgically implanted, which occasionallyinterfered with normal limb function; skin discoloration in the inguinalregion; decreased activity; inappetance; and in the highest-dosagegroup, skin cold to the touch.

To determine the cause of the inflammation and hind limb swelling, aseries of 72-hour continuous infusion investigative studies wereconducted over the following 6 months. The results of those studiesshowed that CXL-1020, when administered in a pH 4 formulation of a 1:1molar ratio of CXL-1020:CAPTISOL®, diluted into a solution of 5%dextrose in water, caused clinical pathology changes consistent with aninflammatory process at dose rates greater than or equal to 0.03μg/kg/min in dogs. Vascular inflammation was observed around the site ofinsertion of the catheter into the femoral vein (15 cm upstream from thecatheter tip), at the catheter tip, and downstream from the cathetertip. The first site of inflammation, the catheter insertion site, causedthe dog hind limb swelling and inflammation observed in theearly-terminated follow-up study. Increasing infusate pH from 4 to 6decreased inflammation, improving the inflammatory profile byapproximately 3-fold. However, significant undesirable side effects werestill demonstrated when CXL-1020 was administered at dose rates greaterthan or equal to 3 μg/kg/min in the dogs.

To avoid the catheter insertion site-associated side effects and toassess whether the vascular inflammation was due to the design of theimplanted catheter, a 24-hour continuous infusion study was conducted indogs using a percutaneous catheter placed in a peripheral (cephalic)vein. After 6 hours of infusion, significant edema was observed in theupper forelimb, downstream from the catheter tip. After 24 hours ofinfusion, clinical pathology changes similar to those observed inprevious studies using an implanted central catheter were detected. Alsodetected was microscopic pathology demonstrating a severethrombophlebitis at the catheter tip and progressing with a gradient oflessening severity downstream from the catheter tip.

To determine whether a local phlebitis would occur in humans upon longerduration dosing, a longer duration study was conducted in healthyvolunteers. The longer duration study included a dose escalation studyin which cohorts of 10 volunteers were to be sequentially administered a24-hour continuous infusion of CXL-1020 at the dose rates of 10, 20, and30 μg/kg/min with a safety assessment between each cohort. Each cohortconsisted of 2 placebo and 8 active treatments with a sentinel pair of 1active and 1 placebo followed by the main group of 1 placebo and 7active treatments. The infusion was via a percutaneous catheter insertedinto a forearm vein. The catheter was switched to the contralateral armafter 12 hours of infusion. The dose rate of 10 μg/kg/min for 24-hourswas found to be well tolerated. In the second cohort, administered adose of 20 μg/kg/min for 24-hours, there were no adverse findings in the2 placebo-treated volunteers but there were mild findings (eitherclinical signs and/or changes in clinical pathology) in all 8 subjectsconsistent with infusion site phlebitis. Based on these results, thelonger duration safety study was halted.

Exploratory studies were continued to determine the cause of theundesirable side effects of CXL-1020 at the higher, but still clinicallydesirable, doses. Studies conducted with the byproduct of CXL-1020, themoiety that remains after nitroxyl donation, was negative, indicatingthat the CXL-1020's side effects were attributable to either the parentcompound, CXL-1020, or to the HNO produced therefrom. Studies wereconducted with alternative nitroxyl donors that were structurallyunrelated to CXL-1020 but had similar half-lives for nitroxyl donation(half-lives of about 2 minutes). For these donors, nitroxyl was at itshighest intravascular concentration at the catheter tip and immediatelydownstream in the vein into which the catheter had been inserted. In allinstances, local vascular side effects at the catheter tip wereobserved. These results suggested that the inflammation was caused bynitroxyl that was rapidly released from the short half-life nitroxyldonors.

5.7.2 Example 9: Compounds of the Disclosure Possess an ImprovedToxicological Profile Relative to CXL-1020

Studies were conducted in male and female beagle dogs. Animals wereallowed free access to drinking water and a commercial canine diet understandard laboratory conditions. Animals were fasted prior to bloodsample collections when indicated by the study protocol. Fluorescentlighting was provided via an automatic timer for approximately 12 hoursper day. On occasion, the dark cycle was interrupted intermittently dueto study-related activities. Temperature and humidity were monitored andrecorded daily and maintained to the maximum extent possible between 64°F. to 84° F. and 30% to 70%, respectively. The dogs were acclimated fora period of at least 1 week. During this period, the animals wereweighed weekly and observed with respect to general health and any signsof disease. The animals were acclimated to wearing a jacket for at leastthree days prior to dose administration. Additionally, the animals werealso acclimated to wearing an Elizabethan collar (e-collar) during thejacket acclimation.

Surgical Procedure and Dosing Procedure

Animals were catheterized the day prior to dose administration. Apercutaneous catheter was placed (using aseptic technique and sterilebandaging) in the cephalic vein distal to the elbow. The animals werefree-moving in their cages during continuous infusion doseadministration. To facilitate continuous infusion dose administration,the peripheral catheter was attached to an extension set routedunderneath a canine jacket and then attached to a tether infusionsystem. To prevent the animals from accessing/removing the peripherallyplaced percutaneous catheter, the catheterization site was bandagedusing Vet Wrap and an e-collar was placed on the animals for theduration of the treatment (i.e., the catheterized period). During thepretreatment period, the venous catheter was infused continuously at arate of approximately 2-4 mL/hr with 0.9% sodium chloride for injection,USP (saline) to maintain catheter patency. Prior to dosing, the infusionsystem was pre-filled (slow bolus infusion) with the respective dosingsolution to ensure that dosing began as soon as the infusion pump wasstarted. The infusion line was connected to a reservoir containing thecontrol or test compound and the infusion was started. Test compositionswere infused continuously, at a predetermined constant infusion rate (1or 2 mL/kg/hr), for 24 hours and were compared at molar equivalent doserates.

Clinical Observations, Clinical Pathology, and Microscopic Pathology

A detailed clinical examination of each animal was performed twice dailyand body temperature measurements and blood samples for clinicalpathology were collected from all animals pre-dose and 6 hours, 12hours, 24 hours and 72 hours post start of composition infusion. At thetermination of the study, all animals were euthanized at their schedulednecropsy and complete necropsy examinations were performed. Selectedtissues were collected, fixed and stored for potential futuremicroscopic examination. The cephalic vein containing the infusioncatheter was dissected intact along with the brachial vein and examinedalong its entire length. The location of the catheter tip was marked onthe unfixed specimen. After fixation, the specimen was trimmed andprocessed to slide to provide transverse histologic sectionsrepresenting the catheter tip and surrounding tissues both proximal anddistal to the catheter tip (i.e., 1 cm distal to the catheter tip, atthe catheter tip, and 1, 5, 10, 15, and 20 cm proximal to the cathetertip). Relative to the catheter tip, “proximal” was defined as closer tothe heart and “distal” was defined as further from the heart.

Safety Assessment

Clinical pathology changes consistent with an inflammatory syndrome wereobserved at some dose rates of compounds of formula (1), formula (2) andCXL-1020. Each compound was formulated with CAPTISOL® (7% w/v) insterile water at a pH of 4. The most sensitive biomarkers of theinflammation were: (1) white cell count (WBC, obtained as (number ofwhite blood cells)/μL by multiplying the values in the rightmost portionof FIG. 4 by 103), (2) fibrinogen concentration (given in mg/dL in therightmost portion of FIG. 4), and (3) C-Reactive Protein (CRP)concentration (given in mg/L in the rightmost portion of FIG. 4). Theseverity of the changes was dependent on the identity of the compoundand the dose rate at which the compound was administered (FIG. 4). InFIG. 4, a score ranging from 0 (low severity) to 2 (high severity) wasassigned to each of these biomarkers of inflammation according to therightmost portion in that figure. A cumulative score was calculated fromthe sum of these marker scores. The NOAELs, determined based on theseclinical pathology markers and expressed in molar equivalent dose rates(μg/kg/min) to CXL-1020, are provided in Table 7.

TABLE 7 No Observed Adverse Effect Levels (NOAEL) of Nitroxyl DonorsNOAEL Compound (μg/kg/min)N-Hydroxy-2-methanesulfonylbenzene-1-sulfonamide <0.03 (CXL-1020)N-Hydroxy-5-methylfuran-2-sulfonamide (1) >20N-Hydroxy-3-methanesulfonylbenzene-1-sulfonamide (2) 3

For CXL-1020, significant elevations in WBC, fibrinogen and CRP wereobserved, even at concentrations as low as 0.03 μg/kg/min. The compoundof formula (1) and the compound of formula (2) each have a NOAEL atdoses significantly higher than that of CXL-1020. The compound offormula (1) has the most favorable toxicological profile, showing noadverse effects at doses at least as high as 20 μg/kg/min. Thisrepresents greater than a 660-fold improvement relative to CXL-1020.

Collectively, these findings suggest that CXL-1020 infusion causes aninflammatory syndrome, which is substantially reduced with the compoundof formula (1) and the compound of formula (2).

The findings suggested that the undesirable vascular side effectsassociated with CXL-1020 at the catheter tip, downstream of the cathetertip and in certain circumstances, upstream of the catheter tip, were dueto local inflammation caused by nitroxyl release. Moreover, it waspostulated that inflammation can be significantly mitigated at thesesites using longer half-life nitroxyl donors. Confirmation was obtainedthrough evaluating the nitroxyl donors through detailed histopathologyof the vasculature at the site of insertion of into the femoral vein (15cm distal to the catheter tip), along the catheter track to the cathetertip, and past the tip downstream 20 cm. Microscopic pathology findingsof edema, hemorrhage, vascular inflammation and perivascularinflammation were determined at particular dose rates of the nitroxyldonors.

FIG. 5 depicts a “heat-map” showing a composite lab score for themicroscopic pathology findings in which the severity of vascularinflammation, hemorrhage, thrombus and vasculardegeneration/regeneration was scored in sections of the vasculature asdescribed above. Findings of (1) edema, (2) vascular and perivascularinflammation, and (3) hemorrhage were scored (each assigned a valueselected from: 0=within normal limits; 1=minimal; 2=mild; 3=moderate;4=severe) in sections of the vessel beginning 1 cm distal (upstream)from the catheter tip progressing 20 cm proximal (downstream) from thecatheter tip. A composite lab score was calculated from the sum of thesefindings scores. In FIG. 5, the cumulative histology composite lab scoreranges from 0-2 (low severity) to 11-12 (high severity). The severity ofthe microscopic changes and the distance from the catheter tip in whichthey were detected was observed to be dependent on the identity of thenitroxyl donor and the dose rate at which the nitroxyl donor wasadministered. The NOAEL values determined based on these microscopicpathology markers for a series of nitroxyl donors, expressed in molarequivalent dose rates (μg/kg/min) to CXL-1020, are provided in Table 8.

TABLE 8 No Observed Adverse Effect Levels (NOAEL) of Nitroxyl DonorsNOAEL Compound (μg/kg/min)N-Hydroxy-2-methanesulfonylbenzene-1-sulfonamide <3 (CXL-1020))N-Hydroxy-5-methylfuran-2-sulfonamide (1) ≥180N-Hydroxy-3-methanesulfonylbenzene-1-sulfonamide (2) ≥180

The findings presented in Table 8 provide additional evidence that thecompounds of formula (1) and (2) have a substantially improvedtoxicological profile relative to CXL-1020. The vascular side effects atany dose decreased in severity as a function of distance from thecatheter tip, and the severity of such vascular side effects decreasedwith decreasing dose. These findings confirmed a large safety margin forcompounds of formulas (1) and (2), which translate into a substantialtherapeutic index in humans, and suitability for intravenousadministration at therapeutically effective doses and dosage rates.

5.8 Stability of Intravenous Dosing Solutions 5.8.1 Example 10: Compoundof Formula (1)—Dosing Solution Stored at 25° C.

The stability of dosing solutions of the compound of formula (1)prepared from a CAPTISOL® concentrate diluted intocommercially-available IV diluents was assessed at 25° C. over 48 hours,with analysis points at 0, 8, 12, 16, 24, and 48 hours after dilution.Due to the analysis points required, two studies were executed withseparate sets of dosing solutions. The first (group A) encompassed alltime points except that at 16 hours. The second (group B) entailedanalysis at 0 and 16 hours only. The concentrates used to prepare thetwo sets of dosing solutions were prepared from two separate vials ofthe same lot of lyophilized drug product (24 mg/mL compound of formula(1)/30%) CAPTISOL®.

Concentrate Preparation

One vial of lyophilized drug product (24 mg/mL Compound of formula(1)/30% CAPTISOL®, pH 4) was reconstituted with 10 mL of water forinjection (WFI) quality water to prepare each concentrate (for dosingsolution groups A and B). The pH values of the resultant solutions weremeasured, and were determined to be approximately 3.9 for both vials. NopH adjustment was performed. The concentrates were diluted and analyzedby HPLC (XBridge Phenyl Column (Waters); UV absorbance detector at 272nm; mobile phase a step gradient of aqueous acetonitrile containing 0.1%(v/v) formic acid), and both were determined to contain 20-21 mg/mL ofthe compound of formula (1), rather than the nominal value of 24 mg/mL,ostensibly due to contribution of the dissolved API and CAPTISOL® to thetotal solution volume.

Diluent Preparation

Commercially-available potassium acetate and potassium phosphatesolutions were selected for evaluation. Potassium acetate was obtainedcommercially, and a USP potassium phosphate solution was preparedaccording to the Hospira product insert for the commercial product. Eachsolution was diluted to 10 mM in 5% dextrose (D5W) and 2.5% dextrose(D2.5W). Commercially-available D5W was diluted 2-fold with WFI qualitywater to produce the D2.5W solution. The pH of each concentrated anddiluted solution was measured; the results are presented in Table 9.

TABLE 9 Results of pH Measurement of Selected Diluents DiluentConcentration pH Acetate 10 mM in D2.5W 6.2 10 mM in D5W 6.0 Initial(2M) 6.7 Phosphate 10 mM in D2.5W 6.8 10 mM in D5W 6.7 Initial (3M) 6.5

Dosing Solution Preparation

The compound of formula (1) concentrate was diluted volumetrically on a5 mL scale into the 10 mM diluent solutions to achieve concentrations of8, 1, and 0.1 mg/mL of the compound of formula (1), as summarized inTable 10. Each sample was prepared in duplicate. The dextrose content inthe 10% CAPTISOL® solution was reduced to ensure that the dosingsolutions were substantially isotonic. Each solution was stored at 25°C.

TABLE 10 Preparation of Dosing Solutions for Stability EvaluationCompound of Formula (1) Dilution CAPTISOL ® (mg/mL) Diluent factor (%w/v) 8.0 10 mM acetate or phosphate 3  10% in D2.5W 1.0 10 mM acetate orphosphate 24 1.3% 0.1 in D5W 240 0.1%

Sample Analysis

Samples were analyzed upon preparation and after 8, 12, 16, 24, and 48hours of storage at 25° C. The visual appearance of each sample wasnoted, the pH was measured, and each sample was analyzed by HPLC forconcentration and presence of the major degradant, the compound offormula (5), depicted below, formed after the release of the HNO group.

Results

The results of the stability evaluation are presented in Table 11, Table12 and Table 13. The presence of a peak corresponding to the degradant(compound of formula (5)) in a sample is denoted by an “X”.

The results were generally consistent for each duplicate within a pairand between corresponding dosing solutions prepared in groups A and B. Adifference in recovery was observed between duplicates at the 24 and 48hour time points for the samples prepared to contain 0.1 mg/mL of thecompound of formula (1) in phosphate.

Complete recovery (within the accuracy of the HPLC method) and absenceof a detectable amount of a compound of formula (5) peak was maintainedover 48 hours for the samples prepared to 8 mg/mL of the compound offormula (1) in acetate- and phosphate-based diluents. These samplesactually contained approximately 7 mg/mL of the compound of formula (1),consistent with the concentration of 20-21 mg/mL compound of formula (1)in the concentrate. In both diluents, stability was superior in thesamples prepared to 8 mg/mL compound of formula (1) than in the samplesprepared to lower concentrations. Without being bound by theory, thebetter stability of these samples compared to those prepared to lowerconcentrations of the compound of formula (1) may be attributed to thehigher CAPTISOL® concentration (10% in the diluted solutions).

All samples remained clear and colorless over the 48 hours of storage.The pH of all samples decreased over time. The known degradant (compoundof formula (5)) was observed at t0 (immediately following preparation ofthe sample) in all samples prepared to contain 0.1 mg/mL of the compoundof formula (1) and at all subsequent time points in all samples preparedto contain 0.1 mg/mL and 1 mg/mL of the compound of formula (1).

In general, stability decreased with decreasing concentration of thecompound of formula (1). Without being bound by theory, the decreasedstability was likely due to the lower percent CAPTISOL® in the dosingsolutions. The initial extent of degradation (through 16 hours) wassimilar in the samples prepared to contain 0.1 mg/mL of the compound offormula (1) in the acetate- and phosphate-based diluents. However, thestability of the samples prepared to contain 1 mg/mL demonstratedsignificantly better stability in acetate than in phosphate.

TABLE 11 Results of Dosing Solution Stability Evaluation at 25° C.,Percent Recovery Compound of Formula (1) Compound of mg/mL Recovery fromt0 Dosing Formula (1) t0 t0 8 h 12 h 16 h 24 h 48 h Solution DuplicateDiluent mg/mL (group A) (group B) (A) (A) (B) (A) (A) 1 a 10 mM 8.0 6.947.06 101%  102%  101%  102%  101%  b acetate 6.95 7.06 101%  102%  101% 102%  103%  in D2.5W 2 a 10 mM 1.0 0.86 0.85 97% 97% 97% 94% 92% bacetate 0.87 0.84 98% 98% 98% 96% 95% in D5W 3 a 10 mM 0.1 0.10 0.09 81%78% 66% 67% 55% b acetate 0.10 0.09 80% 75% 68% 63% 51% in D5W 4 a 10 mM8.0 6.98 6.79 98% 99% 102%  99% 100%  b phosphate 7.00 6.93 99% 94%100%  100%  100%  in D2.5W 5 a 10 mM 1.0 0.87 0.85 89% 86% 86% 78% 71% bphosphate 0.88 0.85 90% 83% 82% 79% 72% in D5W 6 a 10 mM 0.1 0.10 0.1083% 78% 72% 62% 41% b phosphate 0.10 0.10 79% 72% 68% 50% 32% in D5W

TABLE 12 Results of Dosing Solution Stability Evaluation at 25° C., pHCompound of pH Dosing Formula (1) t0 t0 8 h 12 h 16 h 24 h 48 h SolutionDuplicate Diluent mg/mL (group A) (group B) (A) (A) (B) (A) (A) 1 a 10mM 8.0 5.6 5.5 5.4 5.4 5.4 5.4 5.3 b acetate 5.6 5.5 5.5 5.4 5.4 5.3 5.3in D2.5W 2 a 10 mM 1.0 5.7 5.7 5.6 5.7 5.5 5.5 5.3 b acetate 5.9 5.7 5.75.8 5.5 5.5 5.4 in D5W 3 a 10 mM 0.1 6.1 5.9 5.9 5.9 5.4 5.7 5.7 bacetate 5.8 5.9 5.9 5.9 5.3 5.7 5.5 in D5W 4 a 10 mM 8.0 6.3 6.1 5.9 5.95.6 5.5 5.0 b phosphate 6.3 6.2 5.9 5.8 5.6 5.5 4.7 in D2.5W 5 a 10 mM1.0 6.5 6.6 6.3 6.4 6.2 6.1 5.8 b phosphate 6.6 6.5 6.3 6.4 6.1 6.3 6.0in D5W 6 a 10 mM 0.1 6.8 6.7 6.6 6.6 6.3 6.5 6.4 b phosphate 6.8 6.8 6.56.5 6.2 6.5 6.4 in D5W

TABLE 13 Results of Dosing Solution Stability Evaluation at 25° C. -Measuring Appearance of Compound of Formula (5) Compound of Compound ofFormula (5) Dosing Formula (1) t0 t0 8 h 12 h 16 h 24 h 48 h SolutionDuplicate Diluent mg/mL (group A) (group B) (A) (A) (B) (A) (A) 1 a 10mM 8.0 b acetate in D2.5W 2 a 10 mM 1.0 X X X X X b acetate X X X X X inD5W 3 a 10 mM 0.1 X X X X X X X b acetate X X X X X X X in D5W 4 a 10 mM8.0 b phosphate in D2.5W 5 a 10 mM 1.0 X X X X X b phosphate X X X X Xin D5W 6 a 10 mM 0.1 X X X X X X X b phosphate X X X X X X X in D5W

5.8.2 Example 11: Compound of Formula (1)—Dosing Solution Stored at 2°C.-8° C. Followed by Storage at 25° C.

The stability of dosing solutions of the compound of formula (1)prepared from a CAPTISOL® concentrate diluted into commerciallyavailable IV diluents was prepared as described in Example 10. Thesolutions were assessed at 2° C.-8° C. over 24 hours followed by storageat 25° C. over 48 hours. As shown in Table 14, recoveries of thecompound of formula (1) were generally higher than for the correspondingsamples stored at 25° C. for all dosing solutions (see Table 12 fromprevious example), suggesting improved stability for dosing solutionsprepared and stored at 2° C.-8° C. prior to storage at 25° C.

TABLE 14 Results of Dosing Solution Stability Evaluation at 2° C.-8° C.and 25° C., Percent Recovery Compound of Formula (1) Recovery from t0mg/mL 32 h 36 h 40 h 48 h 72 h Compound of t0 t0 24 h 24 h (A) (A) (B)(A) (A) Formula (1) (group A) (group B) (A) (B) 8 h at 12 h at 16 h at24 h at 48 h at Sample # Diluent mg/mL 2-8° C. 2-8° C. 2-8° C. 2-8° C.25° C. 25° C. 25° C. 25° C. 25° C. 1 10 mM 8.0 7.13 6.91 99% 103% 101% 99% 103%  97% 99% acetate in D2.5W 2 10 mM 1.0 0.89 0.89 99% 100% 98%98% 93% 95% 92% acetate in D5W 3 10 mM 0.1 0.10 0.10 97%  97% 92% 89%67% 82% 73% acetate in D5W 4 10 mM 8.0 7.18 7.08 100%  102% 99% 99%100%  97% 97% phosphate in D2.5W 5 10 mM 1.0 0.89 0.88 99% 101% 95% 93%90% 87% 81% phosphate in D5W 6 10 mM 0.1 0.11 0.10 97%  97% 89% 86% 76%76% 63% phosphate in D5W

5.8.3 Example 12: Compound of Formula (2)—Dosing Solution Stored at 25°C.

A series of dosing solutions of the compound of formula (2) for IVadministration was assessed. The selected concentrate of compound offormula (2), prepared at 30 mg/mL in a vehicle of 30% CAPTISOL® at pH4.0, was evaluated at low, mid, and high concentrations (0.1, 1 and 5mg/mL, respectively) upon dilution into various dosing solutions. Fordilution of the compound of formula (2) to 0.1 and 1 mg/mL, three dosingsolutions were evaluated: (1) D5W, (2) D5W with 5 mM K-phosphate (pH=6),and (3) D5W with 20 mM K-phosphate (pH=6). To maintain iso-osmolalityfor dilutions of the compound of formula (2) to 5 mg/mL, theconcentration of dextrose in the dosing solutions was reduced to 2.5%(w/v). Thus, the dosing solutions evaluated were: (1) D2.5W, (2) D2.5Wwith 5 mM K-phosphate (pH=6), and (3) D2.5W with 20 mM K-phosphate(pH=6).

The potential dosing solutions were assessed for visual appearance, pH,osmolality, and concentration and purity by HPLC (XBridge Phenyl Column(Waters); UV absorbance detector at 272 nm; mobile phase a step gradientof aqueous acetonitrile containing 0.1% (v/v) formic acid) afterapproximately 0, 16, 24, and 48 hours of storage at 25° C. All sampleswere clear, colorless solutions—with the sole exception of 5 mg/mL ofthe compound of formula (2) in D2.5W with 5 mM phosphate which had aclear, light yellow appearance after 48 hours at 25° C. All solutionswere iso-osmotic (290+/−50 mOsm/kg)—with the sole exception of 1 mg/mLof the compound of formula (2) in D5W with 20 mM phosphate which had anosmolality of approximately 350 mOsm/kg. Furthermore, with the soleexception of 5 mg/mL of the compound of formula (2) in D2.5W with 5 mMphosphate, all other dosing solutions sustained the compound of formula(2) at the target concentrations of 0.1, 1 and 5 mg/mL over 48 hours.

In addition, the known degradant, the compound of formula (6), which isdepicted below, formed after release of the active nitroxyl group, wasobserved after 16 hours at 25° C. in small quantities by HPLC in thedosing solutions containing phosphate buffer.

The observed amount of the compound of formula (6) was on the order ofthe limit of detection of the method.

The stability of 5 mg/mL of the compound of formula (2) dosing solutionswas further evaluated as a function of pH and buffer. A concentratedsolution of the compound of formula (2), prepared at 30 mg/mL in avehicle of 30% CAPTISOL® at pH 4.0, was diluted to 5 mg/mL into fourpotential dosing solutions. The four dosing solutions were evaluated:(1) D2.5W, 5 mM K-phosphate (pH=6.0), (2) D2.5W with 5 mM K-citrate(pH=6.0), (3) D2.5W, 5 mM K-citrate (pH=5.0), and (4) D2.5W, 5 mMK-acetate (pH=5.0). All dosing solutions of the compound of formula (2)were iso-osmotic (290+/−50 mOsm/kg). After approximately 24 and 48 hoursof storage at 25° C., the dosing solutions were assessed for visualappearance, pH, and concentration and purity by HPLC. The non-phosphatedosing solutions were clear, colorless and sustained the compound offormula (2) at the target concentration of 5 mg/mL over 48 hours; whileconsistent with the dosing solution screen, the 5 mg/mL compound offormula (2) in D2.5W with 5 mM phosphate (pH 6.0) dosing solution wasclear, light yellow in appearance with only 60% recovery of the compoundof formula (2) after 48 hours. Furthermore, the known degradant, thecompound of formula (6), was observed in small quantities by HPLC in allsamples except 5 mg/mL of the compound of formula (2) in D2.5W, 5 mMcitrate (pH 5.0).

After 7 days of storage at 25° C. the non-phosphate dosing solutionswere still clear and colorless in appearance. The smallest increase inacidity over the 7 days was measured for the 5 mg/mL of the compound offormula (2) in D2.5W, 5 mM citrate pH 6.0 dosing solution, while theD2.5W, 5 mM citrate pH 5.0 dosing solution had the smallest change in pHover the initial 24-48 h. Furthermore, after 14 days of storage at 25°C. the samples with dosing solution containing 5 mM citrate pH 6.0 werestill clear, colorless solutions, while the dosing solutions containingeither 5 mM citrate or 5 mM acetate at pH 5.0 were clear, yellowsolutions. The results are summarized in Table 15.

TABLE 15 Recovery of the Compound of Formula (2) from 5 mg/mL DosingSolutions Time Point Dosing Solution Sample 0 h 24 h 48 h (1). D2.5W, 5mM phosphate, 1 101% 100% 60.7% pH 6.0 2 100% 100% 62.8% (2). D2.5W, 5mM citrate, pH 6.0 1 101% 98.6%  96.7% 2 101% 98.8%  96.5% (3). D2.5W, 5mM citrate, pH 5.0 1 101% 100% 99.1% 2 100% 102% 99.3% (4). D2.5W, 5 mMacetate, pH 5.0 1 95.6%  95.4%  95.4% 2 96.0%  96.8%  94.8%

It will be apparent to those in the art that specific embodiments of thedisclosed subject matter may be directed to one or more of the above-and below-indicated embodiments in any combination.

While the invention has been disclosed in some detail by way ofillustration and example for purposes of clarity of understanding, it isapparent to those in the art that various changes may be made andequivalents may be substituted without departing from the true spiritand scope of the invention. Therefore, the description and examplesshould not be construed as limiting the scope of the invention.

All references, publications, patents, and patent applications disclosedherein are hereby incorporated by reference in their entirety.

What is claimed is:
 1. A method of treating a cardiovascular disease,comprising intravenously administering an effective amount of a compoundof formula (1), or a pharmaceutical composition comprising the compoundof formula (1), to a patient in need thereof, wherein the compound offormula (1) is represented by formula:

and wherein the compound or the pharmaceutical composition isadministered at a dose of at least 1 μg/kg/min.
 2. A method of treatingheart failure, comprising intravenously administering an effectiveamount of a compound of formula (1), or a pharmaceutical compositioncomprising the compound of formula (1), to a patient in need thereof,wherein the compound of formula (1) is represented by formula:

and wherein the compound or the pharmaceutical composition isadministered at a dose of at least 1 μg/kg/min.
 3. The method of claim2, wherein the heart failure is acute decompensated heart failure. 4.The method of claim 2, wherein the compound or the pharmaceuticalcomposition is administered at a dose of at least 2.5 μg/kg/min.
 5. Themethod of claim 4, wherein the heart failure is acute decompensatedheart failure.
 6. The method of claim 2, wherein the compound or thepharmaceutical composition is administered in an amount of at least 5μg/kg/min.
 7. The method of claim 6, wherein the heart failure is acutedecompensated heart failure.
 8. The method of claim 2, wherein thecompound or the pharmaceutical composition is administered at a dose ofat least 7.5 μg/kg/min.
 9. The method of claim 8, wherein the heartfailure is acute decompensated heart failure.
 10. The method of claim 2,wherein the compound or the pharmaceutical composition is administeredat a dose of at least 12 μg/kg/min.
 11. The method of claim 10, whereinthe heart failure is acute decompensated heart failure.
 12. The methodof claim 2, wherein the compound or the pharmaceutical composition isadministered at a dose of no more than about 30 μg/kg/min.
 13. Themethod of claim 12, wherein the heart failure is acute decompensatedheart failure.
 14. The method of claim 2, wherein the compound or thepharmaceutical composition is administered at a dose from about 1μg/kg/min to about 100 μg/kg/min.
 15. The method of claim 2, wherein thecompound or the pharmaceutical composition is administered at a dosefrom about 2.5 μg/kg/min to about 100 μg/kg/min.
 16. The method of claim2, wherein the compound or the pharmaceutical composition isadministered at a dose of at least 15 μg/kg/min.
 17. The method of claim16, wherein the heart failure is acute decompensated heart failure.